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+{"text": "Hydrocarbon-degrading biofilms in the treatment of contaminated groundwaters have received increasing attention due to the role played in the so-called \"biobarriers\". These are bioremediation systems in which a microbial consortium adherent to a solid support is placed across the flow of a contaminated plume, thus promoting biodegradation of the pollutant.Rhodococcus erythropolis and Pseudomonas marginalis were the predominant bacterial species in the microbial consortium.A microbial consortium adherent to pumice granules (biofilm) developed from a toluene-enriched microflora in a mini-scale system, following continuous supply of a mineral medium containing toluene, over a 12-month period. Observation by scanning electron microscopy, together with quantification of the biomass attached to pumice, evidenced the presence of abundant exopolymeric material surrounding the cells in the biofilm. Toluene removal monitored during 12-month operation, reached 99%. Identification of the species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis, revealed that Rhodococcus erythropolis and Pseudomonas marginalis, developed on pumice granules, in a mini-scale apparatus continuously fed with toluene.A structurally complex toluene-degrading biofilm, mainly formed by  More recently, biofilms originating from the indigenous microflora of a contaminated groundwater have received increasing attention due to the possibility to develop arriers\" . These aIndeed, the reactive system of a biobarrier is represented by the biofilm developed on the solid support starting from the autochtonous microbial population of the groundwater, where potentially degrading species are present.In this communication, we report the characterization of a toluene-degrading biofilm developed on pumice granules in two packing mini-columns, employed as a laboratory-scale biobarrier, over a 12-month period. That time was long enough for a mature consortium to establish on a solid support [The main objective of the work was to analyse the structure and composition of the biofilm established on pumice stones in a mini-scale apparatus and to ascertain its degradative capability towards toluene, as a starting point for potential applications of a pumice-bound microbial consortium in bioremediation. Pumice, a rock of volcanic origin very abundant in Southern Italy, was chosen as a support material for its high-surface-area-for-unit-volume and relatively low price.Pumice granules packed mini-columns of two different heights were colonized by a microbial consortium, isolated from a gasoline contaminated groundwater sample and enriched in the presence of toluene as the sole carbon and energy source. Afterwards, the mini-columns  were continuously supplied with a mineral medium containing toluene as the target contaminant, and then sacrificed after 6- and 12-month operation, respectively.Biofilms were characterized with three complementary approaches: observation by scanning electron microscopy, quantification of the attached biomass, and identification of the bacterial species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis. Further, the biodegradative capability of the biofilm developed in column 2 was determined by measuring the efficiency of toluene removal, over a 12-month period.Scanning electron micrographs of Fig. Table The discrepancy between data obtained with the two procedures could be attributed to the presence of a massive exopolymeric material in the 12 month-old biofilm, presumably of polysaccharidic nature and unavoidably weighed when the burning method is employed. The electron microscopy analysis suggests that this material is present both in the upper and lower part of the column; its production is linked to the biofilm maturation occurring in the second 6-month period. In fact, an abundant extracellular material covering the cells was evident by scanning electron microscopy of pumice granules sampled from both the upper and lower part of the column , whereas Pseudomonas marginalis (98% of identity) represented only 10% of the entire consortium. On the contrary, in the enriched culture , Pseudomonas marginalis was the predominant species (86%) and Rhodococcus erythropolis was only 10% of the consortium. Apparently, adhesion to the pumice support promoted the growth of Rhodococcus erythropolis, modifying the initial ratio between the two species.Identification of the species based on 16S rDNA comparative analysis revealed that in both biofilms (column 1 and 2) the majority (85%) of the attached cells was represented by Agrobacterium tumefaciens (98% of identity), Hydrogenophaga palleronii (98% of identity), Chryseobacterium scophtalum (98% of identity), Leucobacter komagatae (98% of identity), Brachybacterium articum (96% of identity), Beta proteobacterium (98% of identity), Microbacterium liquefaciens (99% of identities) and Acinetobacter sp.(98% of identity).Less represented species in the three consortia examined  were identified as vs. the loading rate (LR) applied to column 2 , calculated as the slope of the line obtained plotting the average toluene elimination rate (ER) Toluene oxidation in the presence of nitrate as electron acceptor  may be pRhodococcus erythropolis and Pseudomonas marginalis were the predominant species. Apparently, the two species, once attached to pumice stone, gave rise to a specialised community by the production of exopolymers functioning as biofilm stabilizers [Pseudomonas sp. is the most studied single-species, biofilm-forming bacterium [Rhodococcus sp. have not been investigated so far, though the genus Rhodococcus is present in biofilm reactors [The results obtained in this work demonstrate that a structurally complex toluene-degrading biofilm developed on pumice granules, following their inoculation with a microbial consortium obtained by enrichment of toluene-contaminated water. In the biofilm, bilizers . Pseudomacterium ,7; differeactors  for its reactors .Although the microbiological complexity of the consortium established on pumice deserves further investigation in order to clarify the specific role of each species and its contribution to toluene degradation, the results obtained in this work may be relevant for a final design of a biobarrier in which a cheap support as pumice is used for biofilm formation. In particular, the molecular analysis performed revealed the strong impact of immobilization on the structure of the toluene-degrading community, demonstrating the importance of a molecular approach to characterize microbial biofilms.-1 toluene  as the sole carbon and energy source, sparged with air and incubated at 25\u00b0C for two weeks. After this acclimation period, 25% (v/v) of the culture was transferred into fresh MSM medium containing the same toluene concentration and incubated in the same conditions. To allow culture enrichment, the transfer was repeated 10 times, until a biomass (dry weight determination after centrifugation at 3000 g and washing of the collected cells) of 1.2 mg d. w. ml-1 was achieved.A microbial consortium was obtained following enrichment of a gasoline contaminated groundwater sample and subsequently used to inoculate the packing material in the columns. The enrichment was carried out in 500 ml-flasks, adding 100 ml of groundwater to 100 ml of minimal salt medium (MSM), containing high sulphate and nitrate concentrations [-1), respectively previously washed with distilled water, autoclaved  and then soaked in MSM medium containing 20 mg l-1 toluene. The ratio between column and particle diameter was ~50 to reduce wall effects and preferred channelling; the column void volume was 60%. The columns were provided with teflon supports, tubes and connections to prevent abiotic removal of toluene.To study the biofilm development, two glass columns (2.5 cm in diameter and 1 or 3 cm in height), were packed with pumice granules  for one week. After this period, the two columns, henceforth referred as column 1 (2.5 \u00d7 1 cm) and column 2 (2.5 \u00d7 3 cm), were continuously fed upwards at a flow rate (Q) of 0.026 \u00b1 0.003 l h-1, with MSM plus toluene for 6 and 12 months, respectively, and then sacrificed. Operations were carried out at 25\u00b0C. MSM was continuously sparged with air and mixed with the solution containing toluene just before entering the columns. The set-up for the continuous operation of the two minicolumns is schematically presented in Fig. For each column, the attachment of microbial cells to the support material was carried out by recirculating the enriched culture at a low flow rate  was changed step-wise every 4 weeks, from an initial concentration of 0.77 \u00b1 0.03 up to 4.42 \u00b1 1.21 mg l-1, allowing two weeks to achieve culture acclimation before conducting the biodegradation assays in the following two weeks. A total of six values of toluene concentration in the inlet were established, being the highest inlet concentration achieved during the sixth month of operation; then, column 1 was sacrificed, and column 2 was fed with 4.42 \u00b1 1.21 mg l-1 toluene during the remaining 6 months, in order to permit biofilm maturation under constant feeding conditions.The inlet concentration of toluene (Si) and in the outlet (Se) were employed to calculate toluene loading rate (LR) and elimination rate (ER) according to the following equations: ER = Q (Si-Se) / V and LR = Si\u00b7Q/V, where, Q is the inlet feed flow rate (0.026 l h-1) and V is the total volume (0.015 l) of the column, respectively.In the case of column 2, values of toluene concentration in the inlet (SToluene concentration in the gas and liquid phase was determined by the headspace method on a gas chromatographic system (HP 6890 Series) equipped with flame ionization detector (FID), calculating the area of the chromatographic peaks. Toluene concentration values in the outlet of column 2 were the average of at least 10 determinations, carried out either after culture acclimation during the first 6-month operation, or every week during the following 6-month period, when column 2 was maintained at the highest LR. Neither evidence of abnormal biomass growth nor loss of mechanical properties of the supports were recorded during the entire time course of the experiment.-1 NaCl for two hours at 4\u00b0C, processed according to [To perform scanning electron microscopy analysis, pumice granules collected from both columns 1 and 2, together with not colonised granules were fixed with 2.5 % glutaraldehyde in 15 g lThe biomass attached to the support was estimated as volatile solids (VS), after drying at 105\u00b0C and then burning at 600\u00b0C of the colonised pumice granules, collected from both columns. The difference in mass between the dried and the burned samples was the weight of VS . Biomass was also indirectly determined by total protein assay  after detachment of the cells from the support by bead-beating in a homogenizer  and their lysis (60\u00b0C for 90 min with 0.6% w/v SDS), assuming that the average protein content in bacteria is about 50% of cell dry weight . Since cTo identify the microbial species in the biofilm of columns 1 and 2, cells removed from the pumice granules by bead-beating (see above) were plated on TSA (Tryptone Soy Agar). An average of 100 colonies were isolated after serial dilutions from each consortium, and cultured individually in Tryptone Soy Broth (TSB). In parallel, to identify the microbial species present in the enriched culture employed to inoculate the packing material, an average of 100 colonies from the enriched culture were isolated on TSA and cultured individually in TSB.B. subtilis rrnE. These primers are normally utilized as universal primers for eubacteria. After PCR amplification, 1.5 Kbp 16S ribosomal DNA fragments were purified from agarose gels and sequenced. Ribosomal DNA gene sequences of the isolates where compared by BLAST program with those present in the DNA GenBank [Chromosomal DNA was extracted from cells of the isolated strains according to standard methods for bacteria  and used GenBank .DLA:carried out biofilm characterization. VM:carried out the identification of species and helped to draft the manuscript. PP: partecipated in the design of the study. VR: partecipated in the design of the study and setting up of laboratory apparatus. BA: performed toluene analysis. SP: partecipated in the setting up of laboratory apparatus. SR: partecipated in the design of the study and helped to draft the manuscript. DAE: conceived of the study, and partecipated in its design and coordination, drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Caffeine consumption has been reported to decrease bone mineral density (BMD), increase the risk of hip fracture, and negatively influence calcium retention. In this study, we investigated the influence of caffeine on the osteoblasts behaviour.2 (PGE2) and total protein were performed at day 1, 3, and 7. DNA degradation analysis under the caffeine influence was also performed.Osteoblasts derived from newborn Wistar-rat calvaria was used in this study. The effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay. Alkaline phosphatase (ALP) staining, von Kossa staining and biochemical parameters including ALP, lactate dehydrogenase (LDH), prostaglandin E2 content decreased significantly, the LDH and PGE2 secreted into the medium increased significantly. The activation of an irreversible commitment to cell death by caffeine was clearly demonstrated by DNA ladder staining.The results showed that the viability of the osteoblasts, the formation of ALP positive staining colonies and mineralization nodules formation in the osteoblasts cultures decreased significantly in the presence of 10 mM caffeine. The intracellular LDH, ALP and PGEIn summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis. Caffeine and the related methyl xanthines are widely distributed in plants throughout the world. All stable indigenous cultures having access to these plant products have developed drinks containing these stimulants. Thus caffeine is probably the most commonly consumed pharmacologically active compound in the world, certainly in Europe and North America. Caffeine-containing beverage consumption has been reported to be associated with reduced bone mass and increased fracture risk in some observational studies. In 1982, Heaney and Recker's publication first showed a negative effect of caffeine on the calcium economy .The role of caffeine as a risk factor for bone loss is still controversial. Caffeine consumption has been reported to decrease bone mineral density (BMD) , increasThere are four probable ways an agent may increase the fracture risk and/or skeletal fragility of an elder people : (1) an The powder of caffeine  were purchased and diluted in phosphate buffered solution . In the first part of this study, the effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay as described below. Seven different concentrations  were tested for 1 day, 3 days, 7 days and 14 days period.2. The resulting cell suspension was then passed and centrifuged at 1500 rpm for five minutes to pellet the cells. The supernatant was removed and the pellet re-suspended in \u03b1-minimal essential media  as described below. Unambiguous identification of cell populations as osteoblasts is complex and none of the parameters used for defining osteoblasts-like cells are unique to this cell types. The presence of alkaline phosphatase, an early marker of osteoblasts . The cell lysates were then incubated at 37\u00b0C for 2 h. The genomic DNA was extracted by two separations, with phenol/chloroform and then with chloroform only. The DNA pellet was then washed in 70% ethanol and resuspended in 1 mM EDTA, 10 mM Tris-HCl (pH 8.0) at a final concentration of 20 \u03bcg/ml. The DNA fragmentation analysis was performed using a 1.5% agarose gel in 1 mM EDTA, 40 mM Tris acetate (pH 7.6) to visualize the laddering of the samples.For the DNA fragmentation, a concentration of 1 \u00d7 10th day's culture, decreased osteoblasts activities were observed in the presence of various concentrations of caffeine. We selected the 10 mM concentration of caffeine for the further biochemical study because the osteoblasts showed the highest activities during the 3rd and 7th testing period  that led to cell death in the first day's culture; while in the concentrations of 0.5, 1.0 or 2.5 nM caffeine, the appearance of DAN fragmentation appeared at the 3Coffee is one of the most widely consumed psychoactive beverages throughout the world. Many investigators have demonstrated that caffeine, one of the main constituents of coffee, has a variety of pharmacological and cellular responses in the biological systems . These ith day's culture  is aimed at the removal of redundant, misplaced, or damaged cells, or is activated in defense against infected or mutated cells, preventing further proliferation of a pathogen or disease. The process is characterized by morphological changes, including condensation of the nuclear chromatin, DNA fragmentation, cellular shrinkage, and the formation of apoptotic bodies, which are membrane-bound cellular constituents . In animIn summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis."}
+{"text": "Smoking is highly addictive, and nicotine abstinence is associated with withdrawal syndrome in hospitalized patients. In this study, we aimed to evaluate the impact of sudden nicotine abstinence on the development of agitation and delirium, and on morbidities and outcomes in critically ill patients who required respiratory support, either noninvasive ventilation or intubation, and mechanical ventilation.We conducted a prospective, observational study in two intensive care units (ICUs). The 144 consecutive patients admitted to ICUs and requiring mechanical ventilation for >48 hours were included. Smoking status was assessed at ICU admission by using the Fagerstr\u00f6m Test of Nicotine Dependence (FTND). Agitation, with the Sedation-Agitation Scale (SAS), and delirium, with the Intensive Care Delirium Screening Checklist (ICDSC), were tested twice daily during the ICU stay. Agitation and delirium were defined by SAS >4 and ICDSC >4, respectively. Nosocomial complications and outcomes were evaluated.n = 44) were younger and more frequently male and were more likely to have a history of alcoholism and to have septic shock as the reason for ICU admission than were nonsmokers. The incidence of agitation, but not delirium, increased significantly in the smoker group . Nicotine abstinence was associated with higher incidences of self-removal of tubes and catheters, and with more interventions, including the need for supplemental sedatives, analgesics, neuroleptics, and physical restraints. Sedation-free days, ventilator-free days, length of stay, and mortality in ICUs did not differ between groups. Multivariate analysis identified active smoking  as an independent risk factor for agitation. Based on a subgroup of 56 patients, analysis of 28 pairs of patients (smokers and nonsmokers in a 1:1 ratio) matched for age, gender, and alcoholism status found similar results regarding the role of nicotine withdrawal in increasing the risk of agitation during an ICU stay.Smokers (Nicotine withdrawal was associated with agitation and higher morbidities in critically ill patients. These results suggest the need to look specifically at those patients with tobacco dependency by using the FTND in ICU settings. Identifying patients at risk of behavioral disorders may lead to earlier interventions in routine clinical practice. Cigarette smoking is the main addiction in the world . TobaccoBehavioral disorders such as delirium and agitation in the critically ill occur with a high frequency, ranging from 15% to 80% of patients, and have been associated with increased morbidity and risk of mortality -13. ManyThus, we aimed to evaluate the nicotine-withdrawal syndrome in critically ill patients. We hypothesized that dependent smokers may have increased risk for agitation and delirium, and then increased morbidity, such as infections and accidental self-removal of tubes and catheters, related to these behavioral disorders.This prospective observational study was conducted over a period running from June 2007 to April 2008 in two adult ICUs . All patients admitted to the ICUs and mechanically ventilated with either noninvasive ventilation or intubation for respiratory support for longer than 48 hours were considered eligible for the investigation. Patients were excluded if they were younger than 18 years or were determined to have a history of chronic dementia and psychosis, or acute neurologic diseases on admission, such as severe traumatic brain injury, ischemic stroke, or cerebral hemorrhage. NRT was forbidden during the study period. The study was approved by the local ethics committee. Patients were included after informed consent of the patient or next-of-kin was obtained.The following demographic and clinical data were collected at ICU admission: age, gender, medical or surgical origin referring to the primary admission diagnosis, history of hypertension, chronic alcoholism and psychotropic therapy, smoking status, and primary diagnosis on admission to the ICUs. Alcohol consumption was considered chronic if it persisted for the whole year before admission, as defined by the National Institute on Alcohol Abuse and Alcoholism criteria for unhealthy alcohol use in the United States ,16. To aTabagism was evaluated according to the tobacco load, which is quantified in pack-years, and the nicotine dependence, as assessed by the Fagerstr\u00f6m Test of Nicotine dependence (FTND)  Additio, obtaineDiagnostic and Statistical Manual of Mental Disorders (DSM) IV criteria [Agitation was assessed twice daily by nurses or physicians until ICU discharge, by using the modified Sedation-Agitation Scale (SAS)  additio. SAS liscriteria  and feat3 colony-forming units (CFUs)/ml; (2) colonization of central venous catheters: at least one organism at a concentration \u2265103 CFUs/ml identified by culture of the catheter tip with the Brun-Buisson technique [4/ml with the presence of an organism at a concentration of 105 CFU/ml; (4) bacteremia: a positive hemoculture with the isolation of an organism or at least two positive hemocultures for a coagulase-negative Staphylococcus, according to the usual definitions [Nosocomial infections were defined as follows: (1) ventilator-acquired pneumonia: clinical suspicion of pneumonia , and at least one organism isolated by protective specimen brush at a concentration \u226510echnique ; (3) uriinitions .Primary end points were either one or more agitation or delirium events during the ICU stay. Secondary end points were ventilator-free days ; total dose of sedatives and analgesics administered during the ICU stay, including the extra doses needed to abort the episodes of agitation/delirium; and sedation-free days ; and complications related to agitation and delirium, such as requirement for supplemental sedation or physical restraints, use of additional antipsychotic medication; and self-extubation, self-removal of arterial, central venous and bladder catheters, nasogastric tubes, and nosocomial infections.2 test for categoric variables with continuity correction when appropriate, the Fisher's Exact test for proportions, and the Mann-Whitney test for quantitative variables. To determine the set of independent predictors of agitation, a multivariate logistic regression analysis was performed by using backward stepwise selection. In the multivariate model, the outcome variable studied was agitation, defined as success if agitation occurred at least once. Variables with a P value < 0.1 in univariate analysis were included in the regression analysis, and then a P value of 0.2 was used to remove variables from the model. To avoid analyses that might have resulted in biased conclusions because of redundancy of the included variables, SAPS II and SOFA scores were not included in the same model. The performance of the final model on the test set was assessed by using the c-index and its 95% CI. To ensure that our findings were robust, we also performed a case-control approach nested in our study to assess the relative risk (RR) of agitation, according to the smoking status. We matched each smoker with each nonsmoker in a 1:1 ratio. Each pair had to fulfill three conditions: same age (\u00b1 5 years), same gender, and same status regarding alcohol consumption . We analyzed this subset of patients by using the Wilcoxon test, the MacNemar \u03c72 test, and the pair-matched Mantel-Haenszel adjusted RR. Analysis was performed by using MedCalcSS version10.1 . The two-tailed significance level was set at P < 0.05.With a 30% baseline incidence of smokers in the general population, we calculated that a sample size of at least 134 patients was necessary to achieve a 30% absolute difference in agitation/delirium incidence among smokers and nonsmokers at a beta error of 0.2 and an alpha error of 0.05. For univariate analysis, we used the \u03c7P = 0.62, respectively), and in these cases, we have recorded the patient's test responses for analysis. Thus, only six (14%) FTND scores were obtained exclusively from next-of-kin, and were recorded as well.In total, 916 admissions were screened for enrollment. One hundred fifty consecutive patients met the inclusion criteria. Six patients were secondarily excluded, leaving 144 patients for analysis  patients in the overall cohort received noninvasive ventilation. In this subgroup of patients, the rate of intubation was 62% related to failure of noninvasive ventilation. Thus, only 17 patients (five smokers and 12 nonsmokers) were treated exclusively with noninvasive ventilation. Based on SAS and ICDSC scores, no statistical difference was recorded in the number of agitation events between both groups (data not shown).P = 0.0005), but delirium was not affected by smoking status. There was no central effect in the incidence of agitation. In a subgroup analysis comparing weak and strong tobacco dependencies according to FTND, no statistical difference was observed for the development of agitation . Similarly, tobacco-load distribution did not differ between smokers with and without agitation during the ICU stay. In patients in whom agitation developed, the median number of days with agitation events was greater for smokers than for nonsmokers. The rates of accidental removal of tubes and catheters were higher for smokers, as well as the need for supplemental sedative and analgesic medications, physical restraints, and neuroleptics in comparison with nonsmokers  , and then was not identified as independent predictor for agitation in the multivariate analysis.By univariate analysis, the risk factors associated with agitation were age, male gender, SOFA score, and active smoking Table . After a2 = 8.2; P = 0.004), and tobacco dependency was associated with an increased risk for agitation compared with a no-smoking history .Matching was possible in 62 patients representing 43% of the patients considered in the main analysis  ,38-40. HSome study limitations must be addressed. First, objective measurement of tobacco addiction, like urinary cotinine, was not performed, and conclusions drawn from this observational study in a small number of patients cannot be generalized. The lack of any association between the level of addiction and either agitation rate or most important outcomes  may also be the result of the small sample size. Second, tobacco-smoking status and end points were not collected in a blinded manner by nurses and staff, and this point may be considered a potential bias. Third, the use of sedative and analgesic drugs, as well as discontinuation of physical restraints, were left to the attending physician's judgment, and thus, were likely to be somewhat random. As a consequence, \"organ failure\" of the brain (delirium and agitation) may have been over- or underestimated in some cases. And fourth, adjusted analyses can potentially yield distorted associations by selection or omission of variables that influence outcome. Regarding age, gender, alcoholism, and septic shock, a large imbalance between smokers and nonsmokers was observed in our cohort study and may be considered as potential bias. These confounding factors are, however, taken into account in the matched analysis that confirms our finding.In mechanically ventilated patients, sudden nicotine abstinence was associated with severe agitation and its consequences, such as self-removal of tubes and catheters. These results suggest the need to be aware of nicotine-withdrawal syndrome in critically ill patients, and then support the necessity to improve strategies to prevent and treat agitation earlier. Based on these findings, the use of nicotine-replacement therapy should be tested by a well-designed, randomized controlled clinical trial in the ICU setting.\u2022 Agitation is a common event in critically ill patients.\u2022 Nicotine withdrawal was identified as risk factor of agitation, but not delirium, in multivariate and matched case-control analyses adjusted for confounding factors.\u2022 Agitation was associated with a higher adverse-event rate, such as accidental self-removal of tubes and catheters, and new interventions including supplemental sedation and physical restraint.\u2022 These results suggest the need to be aware of nicotine-withdrawal syndrome in critically ill patients, and then to support the necessity to improve strategies to prevent and treat agitation earlier.The authors declare that they have no competing interests.OL and DdC initiated the study and the design. DdC was involved in the interpretation of the results and wrote the manuscript. OL helped to draft the manuscript. AS, CD, MR, NT, PB and PC contributed to the conception of the study. All authors read and approved the final manuscript.This work was presented in part at the 37th Congress of the Soci\u00e9t\u00e9 de R\u00e9animation de Langue Fran\u00e7aise (SRLF) held in January 2009 in Paris, France.Fagerstr\u00f6m Test for Nicotine Dependence (FTND).Click here for fileRiker Sedation-Agitation Scale (SAS).Click here for fileIntensive Care Delirium Screening Checklist (ICDSC).Click here for file"}
+{"text": "Objective. We aimed to investigate the amounts of nitrate, nitrite, and total organic carbon (TOC) in two drinking water sources and their relationship with some gastrointestinal diseases. Methods. This cross-sectional study was conducted in 2012 in Iran. Two wells located in residential areas were selected for sampling and measuring the TOC, nitrate (NO3\u2212), and nitrite (NO2\u2212). This water is used for drinking as well as for industrial and agricultural consumption. Nitrate and nitrite concentrations of water samples were analyzed using DR 5000 spectrophotometer. The information of patients was collected from the records of the main referral hospital of the region for gastrointestinal diseases. Results. In both areas under study, the mean water nitrate and nitrite concentrations were higher in July than in other months. The mean TOC concentrations in areas 1 and 2 were 2.29 \u00b1 0.012 and 2.03 \u00b1 0.309, respectively. Pollutant concentration and gastrointestinal disease did not show any significant relationship (P > 0.05). Conclusion. Although we did not document significant association of nitrite, nitrate, and TOC content of water with gastrointestinal diseases, it should be considered that such health hazards may develop over time, and the quality of water content should be controlled to prevent different diseases. Nitrate is considered as the most prevalent chemical contaminant in the world's groundwater. Organic and inorganic sources of nitrogen are converted to nitrate. After reducing, nitrate can be biologically transformed to nitrogen gas. The growing contamination of public and private well drinking water by nitrate is mostly because of the widespread use of commercial fertilizers and waste .Groundwater is used for agricultural and industrial consumption as well as for drinking water. Humans have altered the nitrogen cycle dramatically over the last decades, and as a result, nitrate is increasingly accumulating in water resources. Globally, human nitrogen production has increased significantly since 1950 due to the use of nitrogen fertilizers. In agricultural areas, groundwater and private and low depth wells have higher levels of nitrate. Fertilizers are most important contributing factor in agricultural areas; however, nitrogen from human waste seems to be an essential source in urban areas with deficient centralized water and sanitation systems .Nitrate is in solution form and is mobile. It could become spread in groundwater and is one of the most common pollutant and concern for human health. It may have several health hazards. Drinking water contamination with nitrate could increase cancer risk, because nitrate could reduce to nitrite and the following nitrosation reactions give rise to N-nitroso compounds, which are especially carcinogenic and can act systemically. The suggested regulatory limit for nitrate is too conservative. A symposium in 2004 on drinking water nitrate and health assessed nitrate exposures and related health effects about the current regulatory limit. The contribution of drinking water nitrate toward endogenous formation of N-nitroso compounds was evaluated with a focus toward identifying subpopulations with increased rates of nitrosation , 4.Drinking nitrate and nitrite under conditions likely to form N-nitroso compounds (NOCs), called endogenous nitrosation, is considered possibly carcinogenic to humans. Nitrate in drinking water is completely related to urine nitrate levels as well as excretion of nitrosoproline, a biomarker of endogenous nitrosation .Water with high nitrate concentration is not suitable for human consumption, especially when its concentration exceeded the threshold limit 50\u2009mg/L) recommended by the health authorities such as the World Health Organization (WHO) [0\u2009mg/L reHuman alteration of the nitrogen cycle has resulted in continuing accumulation of nitrate in the water resources. According to recent studies, a significant relationship exists between nitrate in drinking water and cancer types , 5\u20137.Chemical analysis of a study in Iran showed that groundwater and water supply have considerably high levels of nitrate (from 52.3\u2009mg/L to 52.69\u2009mg/L). Nitrate level in some underground water wells in the landfill area was over the drinking water standard (50\u2009mg per liter) , 9.Growing body of evidence proposes the relationship between nitrate level in drinking water and gastric cancer \u201312.This study aimed to investigate the amounts of nitrate, nitrite, and TOC in two drinking water sources and the relationship of nitrate and nitrite levels with some gastrointestinal diseases.This cross-sectional study was conducted from Feb to June 2012 in Shahrekord city in central part of Iran. We selected this city because the level of nitrate in drinking water wells is high in this area with many underground water sources including approximately 493 deep wells, 321 wells, 171 deep semisubterranean, and 93 springs.\u2212), and nitrite (NO2\u2212). This water is used for drinking as well as for industrial and agricultural consumption.Two wells located in residential areas were selected for sampling and measuring TOC, nitrate  and six samples (three samples per well) for TOC test.For sampling, we used one-liter plastic containers and filled each bottle with water of wells; then we emptied out their contents and once more about 900\u2009mL of water was filled up to provide enough space for better shaking and mixing.Samples were delivered to the Chemistry Laboratory of Health Faculty, Isfahan University of Medical Sciences, in less than 3 hours, and then they were analyzed after 24 hours.Nitrate and nitrite concentrations of samples were read and analyzed using DR 5000 spectrophotometer with a wavelength of 220\u2013275\u2009nm. Experiments were repeated two times for each sample.The spectrophotometer uses indicator compounds to determine the concentration of nitrate in samples. The HACH Test kit was used; it involves mixing a premeasured amount of the indicator compound with a 50\u2009mL of sample water; then the absorption in a particular wavelength was measured. After mixing and a delay for reaction, a color appeared (for nitrogen it is amber color), the darkness of which was measured in the spectrophotometer. The sample was introduced to the spectrophotometer via a pour-through-cell apparatus to minimize optical interferences from sample handling.The program offered by the company (HACH 357 N Nitrate UV) was selected from the menu on the device. The device can be calibrated with distilled water. 50\u2009mL of sample prepared with 1\u2009mL of 1\u2009N hydrochloric acid and the prepared sample was transferred to the quartz cell. Thereafter, the absorbance was displayed and we read it.Measuring nitrite schedule was provided by the spectrophotometer (371\u2009N Nitrite LR PP). This device was calibrated with samples of water, then we added a package of NitriVer 3 Nitrite reagent to 10\u2009mL water samples, and after 20 minutes we read the absorption rate.For measuring TOC, samples were transferred to laboratory of Isfahan Water and Sewage Company.The information of patients was collected from the Medical Records Department of Gastroenterology of Al-Zahra Hospital, Isfahan University of Medical Sciences. Patients with gastrointestinal diseases were referred from Shahrekord to this hospital. We selected this hospital because it is the main referral hospital of the region for gastrointestinal diseases, and patients of neighbor cities, including Shahrekord, are referred to this center.For statistical analysis, we categorized diseases to the following four groups: (1) bile duct diseases; (2) gastric diseases; (3) intestinal diseases; (4) other gastrointestinal diseases.t- and Kruskall Wallis tests. The significance level was set at P < 0.05.Data were analyzed by the Statistical Package for Social Sciences software version 20.0 . Data are presented as mean and standard deviation. Comparison of values in various groups was assessed by One-Sample The results of the analysis of water samples for nitrate, nitrite and TOC measurements are presented in P > 0.05).The mean of TOC concentrations in areas 1 and 2 was 2.29 \u00b1 0.012 and 2.03 \u00b1 0.309, respectively (The frequency of abovementioned diseases according to the codes ((1) bile duct diseases; (2) gastric diseases; (3) intestinal diseases; (4) other gastrointestinal diseases) was as follows: 47.6%, 19%, 19%, and 14.3%.\u2009area 1: 0.25%, 0 %, 50%, and 25%,\u2009area 2: 52.9%, 23.5%, 11.8%, and 11.8%.The corresponding numbers in two sampling areas were as follows:P > 0.05).Evaluation of the relationship between pollutant concentration and gastrointestinal disease did not show any significant relationship in two cities .Some research studies suggest that increasing the allowed levels of nitrate in drinking water can be without risk to human health. Furthermore, some persons with high rates of endogenous formation of carcinogenic N-nitroso compounds may be vulnerable to the development of gastrointestinal cancers. Given the extensive experimental data suggesting a role for nitrate in the formation of carcinogenic N-nitroso compounds and the widespread exposure to nitrate in the population, limited epidemiologic data exist on addressing the possible association of nitrate in drinking water with cancer risk.Nitrate levels in water supplies have been increasing at global level; therefore, additional population-based studies with well-characterized exposures are urgently needed to further our understanding of cancer risk associated with nitrate ingestion  Given thThe major limitation of our investigation is its cross-sectional nature; thus a causal relationship cannot be inferred from our findings, and longitudinal studies are required to test for causality and the clinical importance of our findings.Although we did not document significant association of nitrite, nitrate, and TOC content of water with gastrointestinal diseases, it should be considered that such health hazards may develop over time, and the quality of water content should be extensively controlled to prevent different health hazards."}
+{"text": "Objective. To evaluate trophoblastic cell proliferation and angiogenesis in tubal pregnancy assessed by immunohistochemical study and their correlation with an average variation of \u03b2-hCG in an interval of 48 hours before surgery. Methods. A prospective study was conducted on 18 patients with a diagnosis of tubal pregnancy. The patients were divided into two groups of ectopic pregnancy of which 11 showed rise of \u03b2-hCG levels and 7 patients showed declining \u03b2-hCG levels in an interval of 48 hours prior to surgery. Trophoblastic cell proliferation and angiogenesis were assessed by Ki-67 and VEGF, respectively. Trophoblastic cell proliferation was assessed by Ki-67 and was classified into three groups . The cases analyzed for VEGF were divided into three groups . Statistical analysis was performed using the chi-square, ANOVA, and Kruskal-Wallis tests. Results. The mean variation in the serum \u03b2-hCG levels in 48 hours in tubal pregnancy patients correlated with trophoblastic cell proliferation assessed by Ki-67 and showed a decline of 13.46% in grade I, a rise of 45.99% in grade II, and ascension of 36.68% in grade III (P = 0.030). The average variation in the serum \u03b2-hCG in 48 hours, where angiogenesis was evaluated by VEGF, showed a decline of 18.35% in grade I, a rise of 32.95% in grade II, and ascension of 37.55% in grade III (P = 0.047). Conclusions. Our observations showed a direct correlation of increased levels of serum \u03b2-hCG in 48h period prior to surgery with higher trophoblastic cell proliferation assessed by Ki-67 and angiogenesis assessed by VEGF in tubal pregnancy. An ectopic pregnancy is any pregnancy where the embryo is implanted outside the uterus cavity and tubal pregnancy is when the embryo is implanted in a fallopian tube. Hence, tubal pregnancy is one type of ectopic pregnancy. Ectopic pregnancy (EP) is the leading cause of maternal mortality in the first trimester of pregnancy resulting from acute abdominal bleeding , 2. It iTreatment can be surgical , clinical with methotrexate (MTX), or expectant management. In cases of tubal rupture, the classical treatment is salpingectomy, while conservative surgery is indicated in unruptured tubal pregnancy and is desirable to maintain fertility.The likely cause for the difference in outcome of cases is related to the degree of infiltration of trophoblast in the fallopian tube; thus the study of infiltration modulators allows an understanding of the pathological processes of EP . The immStudies have shown that at least two processes are closely related to trophoblastic infiltration: the first is related to the proliferative capacity of the cells and the second is related to the angiogenic action at the site of implantation \u20138.The proliferative activity of any tissue can be determined by the cell proliferation process resulting from the cascade of events which ensures cell division and replication \u201311. The Vascular endothelial growth factor (VEGF) is produced and secreted under hypoxic  conditio\u03b2-hCG levels in 48-hour period prior to surgery with immunohistochemical analysis using Ki-67 to assess trophoblastic cell proliferation and VEGF to assess angiogenesis in tubal pregnancy.The objective of this study was to correlate the serum We conducted a prospective, descriptive, and observational study of 18 patients with EP from January 2011 to March 2012. This study was approved by the Ethics Committee of the Federal University of S\u00e3o Paulo (UNIFESP) under number 1986/08; signed consent forms were obtained from patients participating in this study.\u03b2-hCG between two measurements in an interval of the onset of tubal pregnancy at the end of 48 hours before surgery. Exclusion criteria were nonrepresentative sample for immunohistochemical analysis and nontubal ectopic pregnancy. The patients enrolled in this study were from a tertiary care hospital of UNIFESP. All patients with unruptured tubal pregnancy on whom two measurements of serum \u03b2-hCG levels were determined in an interval of 48 hours were invited to participate in this study. Some of the invited patients chose not to participate and were referred to nonsurgical treatment ). Thus, the selection of the study participants was random. In this study participants were divided into two groups according to the variations in serum \u03b2-hCG levels. Ascension group was characterized by stable patients with unruptured tubal pregnancy showing no symptoms and presenting rising \u03b2-hCG levels in an interval of 48\u2009h between the two measurements, and the decline group was characterized by stable patients presenting \u03b2-hCG levels declining in an interval of 48\u2009h between the two measurements.Inclusion criterion was diagnosis of unruptured tubal pregnancy with rising or declining levels of \u03b2-hCG levels. All patients initially underwent the noninvasive diagnosis of EP based on TVUS and measurement of \u03b2-hCG. The goal was to take two measurements of serum \u03b2-hCG in an interval of 48\u2009h before surgery. After obtaining the curve of \u03b2-hCG all patients underwent surgery. The diagnosis was confirmed intraoperatively and salpingectomy was performed. An experienced pathologist (JNS) who was blinded to the clinical and laboratory characteristics of patients carried out immunohistochemical analysis. To reduce intraobserver variability all analyses were conducted at a time only in the following ways.Diagnosis of EP was based on the protocol used in the Department of Obstetrics of UNIFESP and is based on an association between the patient's prior clinical history, their gynecological examination, and results of transvaginal ultrasound (TVUS), as well as on the quantitative \u03b2-hCG levels using enzyme immunoassay .Ten (10)\u2009mL of venous blood was collected in a plain tube. Serum separated from the blood was used to confirm the diagnosis of ectopic pregnancy by detecting the increase or decrease in Tissue samples obtained from salpingectomy after fixation in xylene and processing in graded alcohol were embedded in paraffin blocks and sectioned in serial sections of 3-micron thickness and mounted on positively charged slides for immunohistochemical analysis. Sections were deparaffinized in xylene, rehydrated in series of graded alcohol solution, and finally rinsed in water. After blocking the endogenous activity with 3% hydrogen peroxide solution for 15 minutes at room temperature, sections were washed in 1X Tris buffered saline . 1X TBS was used for all the dilution of antibodies and washes during the immunohistochemical staining process. Avidin-biotin peroxidase complex (ABC) immunostaining kit was used, according to manufacturer's protocol .First the proliferative activity of the trophoblastin cut sections was evaluated by using the antibody to cytokeratin 7 and then by using Ki-67 antibody. Immunohistochemical staining for Ki-67 was carried out according to the specification described in the universal peroxidase kit . Positive control used for Ki-67 was tonsil and the negative control was devoid of the primary antibody. Sections were deparaffinized in xylene, rehydrated in series of graded alcohol solution, and finally rinsed in water. Sections were treated with normal serum followed by primary antibody MIB-1 (Immunotech) and stained with hematoxylin. A cell was considered positive when part or all of the nuclei are colored. The nuclei staining analysis divided the cases into three groups: grade I (less than 1/3 of stained nuclei), grade II (from 1/3 to 2/3 of stained nuclei), and grade III (more than 2/3 of the stained nuclei).VEGF expression in the section was carried out using polyclonal anti-VEGF antibody . Positive control used for VEGF was placenta and negative control was devoid of the primary antibody. Slides were analyzed by light microscopy to determine reaction positivity. In this analysis, patients were divided into three groups: grade I: less than 1/3 of the stained cytoplasm, grade II: 1/3 to 2/3 of the stained cytoplasm, and grade III: more than 2/3 of the stained cytoplasm.P value was less than 0.05. The number of cases was based on two previous articles that assessed Ki-67 and EP [Obtained data were summarized using the mean, standard deviation, median, minimum, and maximum values for numeric variables. Data analysis was performed employing the program Minitab Inc., version 1.16, 2010. Statistical comparison between groups I, II, and III on the basis of nuclei staining and VEGF staining and categorical variables was performed using chi-square, ANOVA, and Kruskal-Wallis tests. The result was considered statistically significant when the 7 and EP , 5. We d\u03b2-hCG was 6055.59\u2009mIU/mL on the day of admission (ranging from 415\u2009mIU/mL to 24.590\u2009mIU/mL).The mean age of patients was 27.6 years (range 24\u201340 years), mean gestational age at the time of hospitalization was 7.72 weeks (range 5 weeks and 3 days to 10 weeks), and the overall mean level of \u03b2-hCG levels in an interval of 48\u2009h prior to surgery in patients with trophoblastic cell proliferation assessed by Ki-67 showed a decline of 13.46% in grade I (8 cases), a rise of 45.99% in grade II (7 cases), and a rise of 36.68% in grade III (3 cases) in cell proliferative activity (P = 0.030) (The mean variation of = 0.030) .\u03b2-hCG in 48 hours showed a decline of 18.35% in grade I, a rise of 32.95% in grade II, and ascension of 37.55% in grade III (P = 0.0477)  .\u03b2-hCG levels, greater is the biological activity of the trophoblast. Based on this assumption and trying to understand the growth and activity of trophoblast in tubal wall, we compared the ascension and decline groups in cell proliferation assessed by Ki-67. We found that 10 cases (55.55%) had a mean increase of 41.33% of the \u03b2-hCG levels in 48 hours, with cell proliferation grades II and III, and 8 cases (44.45%) had a mean decrease of 13.46% of the \u03b2-hCG levels in the same period, with grade I cell proliferation, outlining a statistically significant correlation (P = 0.030) between cell proliferation and variation of serum \u03b2-hCG levels in 48\u2009h.With respect to cell proliferation assessed by Ki-67 , 5 showe\u03b2-hCG levels in 48\u2009h, in cases with grade III cell proliferation, demonstrating that an active trophoblast results in elevation in \u03b2-hCG levels within 48\u2009h prior to surgery. Studies have shown that increase in \u03b2-hCG level in 48 hours is associated with failure of systemic treatment with MTX [\u03b2-hCG levels within 48 hours is greater than 36.28% \u00b1 44.31% the therapy with MTX had a higher risk of failure.Klein et al.  and Kisswith MTX \u201314. da Cwith MTX  demonstr\u03b2-hCG levels within 48 hours was greater than 44.45% patients showed trophoblastic cell proliferation activity of grades II and III, assessed by Ki-67, correlating our results with that of da Costa Soares et al. [\u03b2-hCG level within 48 hours before treatment as an indicator for failure of medical treatment with MTX [In our study, we observed that when the variation of s et al. . The Amewith MTX .\u03b2-hCG level is low but show rapid increase in \u03b2-hCG levels in 48 hours, there is greater chance of tubal rupture, demonstrating an active trophoblast. This information should be considered as an option for MTX treatment to avoid persistence of trophoblastic tissue and treatment failure [In cases where initial  failure .\u03b2-hCG levels in 48 hours [\u03b2-hCG level less than 1500\u2009mIU/mL. They observed that in 80 cases the \u03b2-hCG level declined in the second test, whereas in 64 patients the \u03b2-hCG level increased. The success obtained in patients with a decline in \u03b2-hCG was 88.8% and the rise of \u03b2-hCG level was 51.6%. Therefore, the decline of \u03b2-hCG level in 48 hours is predictive of success for expectant management. Data obtained from our study shows that most of the patients with declining \u03b2-hCG levels in 48 hours also showed low trophoblastic cell proliferation assessed by Ki-67, equivalent to grade I (mean decline in \u03b2-hCG levels in 48 hours was 13.46%), which gives better expectant management of EP and does not need surgical treatment.The main inclusion criterion for expectant management of EP is declining 48 hours . Mavrelo48 hours  have sho\u03b2-hCG level, where the peak of the curve, in other words, the time when the \u03b2-hCG levels reach their maximum values, is decisive in the evolution of case because at this point two things can happen: the case can progress to tubal rupture with acute hemorrhage in abdomen or can start the process of involution and reabsorption, evolving to spontaneous healing.EP has a curve evolution with the rise and decline of The trophoblast implantation into the tube occurs in a hypoxic environment; this event triggers the mechanism that stimulates the production of VEGF . Previou\u03b2-hCG level, we performed immunohistochemical analysis comparing this factor with the groups of patients with the rise and decline of \u03b2-hCG level in 48 hours. In the assessment of angiogenesis by VEGF, we observed that patients with the mean rise of 37.55% of \u03b2-hCG levels had increased angiogenesis and patients with mean decline of 18.35% of \u03b2-hCG levels showed low angiogenesis, with a statistical significance (P = 0.0477).In order to understand the correlation between VEGF and Cabar et al.  performe\u03b2-hCG level and the trophoblastic activity. The variation of \u03b2-hCG level in 48 hours was directly related to the trophoblastic cell proliferation assessed by Ki-67, and mean \u03b2-hCG level in 48 hours was related to angiogenesis assessed by VEGF.Despite small sample size, the results of this study reinforce the importance of the correlation between \u03b2-hCG before instituting treatment reflects the degree of cell proliferation, essential condition for clinical indication. The microscopic demonstration of the phenomena observed with clinical experience of conservative treatment is of great value to better selection of cases for this type of treatment in tubal pregnancy avoiding failures and sparing patients the therapy failure.In this research we seek to unravel the immunohistochemical factors associated with failure of conservative treatment of ectopic pregnancy. Relevant information from this study is that the curve of evolution of"}
+{"text": "Tayassu tajacu) has already been determined. This study aimed at investigating and comparing the effects of medetomidine and atipamezole on the seminal variables of collared peccaries undergoing electroejaculation as well as at determining whether these drugs affected the erectile and ejaculatory functions of this species.Alpha adrenergic drugs are usually used in the treatment of erectile and ejaculatory dysfunction in humans. The influence of such drugs on the seminal characteristics of wild animals has not been verified; whereas their impact on the seminal characteristics and erectile and ejaculatory functions of collared peccaries (6 sperm/ml) and MP (220.2\u2009\u00b1\u200949.8 \u00d7 106 sperm/ml); however, both these treatments did not differ from P treatment (180.0\u2009\u00b1\u200950.7 \u00d7 106 sperm/ml). No statistically significant difference was observed among all treatments with regard to erectile function. With regard to ejaculation time, no significant difference was observed between the MP and AP treatments; however, when compared with the P treatment, AP exhibited a significantly higher difference.A statistically significant difference in sperm concentration was observed between AP (100.0\u2009\u00b1\u200926.0 \u00d7 10When collared peccaries were anesthetized with propofol, neither medetomidine nor atipamezole significantly affected the characteristics of the semen or the erectile function, despite the fact that the AP treatment increased ejaculation time. Therefore, the data indicate that using propofol alone is an effective anesthetic protocol for collecting semen in collared peccaries. Other non-injectable anesthetic drugs, such as inhaled anesthetics, may be used in future research to collect semen from peccaries. Tayassu tajacu), which is an endangered species in the Caatinga and Atlantic Forest biomes ..Tayassu Propofol is considered a safe anesthetic for both the induction and maintenance of anesthesia in collared peccaries ,9], and, and9], Several anesthetic agents interfere with the mechanisms that control erectile function, thus emphasizing the need for a careful selection of anesthetics. Opioid analgesics; anticholinergics; anticonvulsants; antihypertensives such as alpha and beta blockers; and tranquilizers such as phenothiazines, butyrophenones, and benzodiazepines may contribute to or even cause erectile dysfunction . Many drSince 1989, studies have been conducted on the collared peccary, and their spermatogenesis has been described . SubsequIn a few studies conducted on electroejaculation in collared peccaries, the semen was tested after electroejaculation to see whether the pre-anesthetic or anesthetic drugs used for sedation, restraint, or anesthesia correlated with differences in sperm quality ,5],18],[18]5],5],. The aimExperimental protocols and animal care were approved by Ethics Committee on Animal Use of UFERSA and were registered at the Chico Mendes Institute for Biodiversity Conservation (ICMBio-IBAMA) (no. 32480\u20131).Twelve sexually mature male collared peccaries aged 21 (\u00b11) months each and weighing 20 (\u00b10.1\u00a0kg) were used. The animals belonged to the Center of Multiplication of Wild Animals at the Universidade Federal Rural do Semi-\u00c1rido, which is located in semiarid northeast Brazil .The males were isolated from the females at three months before the commencement of the study and were maintained under a natural photoperiod (12\u00a0hours/day). They were kept outdoors in paddocks in groups of three animals and were fed a diet consisting of corn (79.8%), soybeans (15.4%), wheat bran (1.45%), calcium (2.6%), vitamins (0.2%), and other minerals (0.05%) complemented by tropical fruits. Water was freely available. Commencing at 20\u00a0hours before the experiments, animals were not allowed to drink water, and at 12\u00a0hours before the experiment, they were also put off feed.On the day of the experiment, the animals were captured and restrained by netting. Then, the cephalic vein was catheterized (22-gauge catheter) and isotonic saline solution was administered at a rate of 5\u00a0ml/kg/min until the end of the electroejaculation procedure. Each animal underwent all three treatments, one at a time, being used as the control for themselves after a 15-day (washout) interval. Twelve repetitions were made for each treatment.The sequence of administration of treatments was random, but twelve repetitions for each treatment were performed. The control treatment, or P treatment, was the one in which the animals received only anesthesia with propofol  at a dose of 5\u00a0mg/kg IV and no pre-anesthesia drug. The second treatment, the MP treatment, consisted of medetomidine  at a dose of 40\u00a0\u03bcg/kg IV and, subsequently, propofol  at a dose of 5\u00a0mg/kg IV. The third treatment, the AP treatment, was a dose of 200\u00a0\u03bcg/kg IV of atipamezole  followed by propofol  at a dose of 5\u00a0mg/kg IV.In the experiments with all protocols, when the animal showed signs of awakening, one-fourth of the induction dosage of propofol was administered in bolus to keep the animal in a superficial anesthetic state as previously reported .The semen samples were collected using an electroejaculator  that was connected to a 12-V source. The stimulatory cycle comprised 10 stimuli at each voltage, from 5\u00a0V up to 12\u00a0V and in increments of 1\u00a0V. Each electrical stimulus lasted for 3\u00a0s with breaks of 2\u00a0s after each stimulus. The stimuli were maintained for a period of 10\u00a0min. The electroejaculator probe containing linear electrodes measured 15\u00a0cm in length and 1.3\u00a0cm in diameter; 12\u00a0cm of the probe were inserted into the rectum of each peccary . The semThe erection was evaluated concurrently with the onset of electroejaculation. The presence or absence of an erection and whether the exposure of the penis was complete or not were recorded.The ejaculation time was measured from the commencement of electroejaculation till the start of ejaculation.The semen volume was measured using micropipettes. The sperm motility (%) and vitality (0\u20135) were immediately assessed using light microscopy under 100x and 400x magnification. The percentage of live spermatozoa was determined by analyzing a slide that was stained with Brome Phenol Blue under light microscopy (400x); 200 cells per slide were counted. After the initial assessment, a 10\u00a0\u03bcL semen aliquot was diluted in 10% buffered formalin (2\u00a0mL) in order to kill and imobilize the spermatozoa, and the sperm concentration was determined using a Neubauer counting chamber. The functional integrity of the sperm membrane was evaluated by a hypoosmotic swelling (HOS) test in which distilled water (0\u00a0mOsm/L) was used as the hypoosmotic solution, and a 0.01\u00a0mL semen aliquot was diluted in a 0.09\u00a0mL hypoosmotic solution and kept in a water bath at 38\u00b0C. After 45\u00a0min, an aliquot of semen was placed on a glass slide, covered by a coverslip, and evaluated by microscopy (400\u00d7), counting 200 cells. Spermatozoa presenting swollen coiled tails were considered as representing a functional sperm membrane .At the end of electroejaculation, the animal was placed in dorsal recumbancy on the table for ventral hair removal and sterile skin preparation of the abdomen for urine sampling. After localizationof the bladder by means of ultrasonography  with an 8\u00a0MHz-frequency microconvex transducer, real-time B-mode, a 16-gauge catheter was introduced into the bladder, aspirating the urine with the aid of a disposable 20\u00a0mL syringe. The urine sample was collected immediately and analyzed by evaluating the presence or absence of sperm cells and the concentration thereof. The cystocentesis was done to verify whether any treatment could promote sperm flow into the urinary bladder of the peccary after electroejaculation.The analyis was performed using SAS statistical software  version 9.0 for Windows and SigmaPlot  version 12.0, and data were expressed as the mean\u2009\u00b1\u2009the standard error. All data were primarily assessed for normality using the Shapiro\u2013Wilk test, and the homogeneity of variance was assessed by the Bartlett test. Whenever necessary, logarithmic transformation was used for determining the parametric assumptions. An arccosine transformation was used in the case of variables expressed in percentages; that is, motility, functional integrity of the sperm membrane, and live spermatozoa. However, in the tables, the results were presented without statistical transformation. The semen parameters were analyzed using a one-way ANOVA for repeated measures, and this was followed by the Tukey test. The evaluation of erection and the time of ejaculation latency were analyzed by Cochran\u2019s Q test and McNemar\u2019s test in accordance with the architecture of the data. For all the statistical analyses, significance was defined as P\u2009<\u20090.05.When the treatments were evaluated for their ability to promote erection, no statistically significant difference was present among any of the treatments Table\u00a0.With regard to ejaculation time, no significant difference was observed between the MP and AP treatments; however, when both these were compared with the P treatment, the AP treatment exhibited a significant difference: The time required for ejaculation with the AP treatment was significantly longer than that for the other treatment Table\u00a0.Variables such as ejaculate volume, sperm motility, vitality, and functional integrity of the membrane did not present significant differences between the three treatments (P\u2009>\u20090.05). Only the sperm concentration exhibited a significant difference between the three treatments (P\u2009>\u20090.05) Table\u00a0. The MP 6 sperm/mL of urine) and the AP treatment (2.3\u2009\u00b1\u20091.8 \u00d7 106 sperm/mL of urine). However, neither of these treatments exhibited a statistically significant difference when compared with the P treatment (5.2\u2009\u00b1\u20093.6 \u00d7 106 sperm/ml of urine).Sperm were present in the urine samples from all three treatments. The sperm concentrations in the urine samples differed between the MP treatment  [Sperm concentration values obtained in this study were higher than those reported in collared peccaries anesthetized with acepromazine/tiletamine/zolazepam (13.8\u2009\u00b1\u20095.7 \u00d7 10perm/mL) . The lowperm/mL) ; hence, Sperm concentrations similar to those reported in this study were obtained (118.0\u2009\u00b1\u2009158.4 \u00d7 106 sperm/mL) by using a propofol-alone protocol . An incrThe difference in sperm concentration between the AP treatment and the MP treatment can be explained by the interaction between the alpha-2 agonist or antagonist drug and the sympathetic nervous system. The sympathomimetic effect is mainly due to the effect of the agonist drug on the alpha-2 postsynaptic receptors as well as on the alpha-1 receptors, although these drugs have a high selectivity for alpha-2 receptors . The actThe penis receives neural input from two sympathetic sources. The hypogastric nerve arises from the caudal mesenteric ganglion (derived from the sympathetic chain at T12-L5) and joins the pelvic nerve to form the pelvic plexus, beyond which the cavernous nerve runs directly to the penis. Other sympathetic fibers run caudally in the sympathetic chain to S1-S3, where they exit with the pudendal and pelvic nerves .In erectile tissue, both alpha-1 and alpha-2 and beta-2 adrenoceptors are expressed, and they probably mediate vasodilation in response to the increase in adrenaline during erection . In esse6 sperm/ml of urine) pre-anesthetized with tiletamine/zolazepam after electroejaculation [The data presented in this study on the concentration of sperm in the urine after electroejaculation are the first published for the collared peccary. The average concentration of sperm observed in the urine for the P treatment in this study is similar to the values found in pigs (3.16\u2009\u00b1\u20099.2 \u00d7 10culation . This reculation . Sperm wculation . In contculation  or medetculation . The samculation . The bloculation . The adrculation .The lack of significant differences between treatments with regard to their ability to affect erection can be explained by the fact that the specific agonist and antagonist alpha-2 adrenergic drugs have little or no role in alpha-1 adrenoceptors. The alpha-2 adrenoceptor expressed in cavernous tissue has little functional activity in the erection . TherefoNo influence on ejaculation latency time was observed either in this study or in others  when an When collared peccaries were anesthetized with propofol, neither medetomidine nor atipamezole significantly affected the characteristics of the semen or the erectile function, despite the fact that the atipamezole treatment increased ejaculation time. Therefore, the data indicate that using propofol alone is an effective anesthetic protocol for collecting semen in collared peccaries. Other non-injectable anesthetic drugs, such as inhaled anesthetics, may be used in future research to collect semen from peccaries.None of the authors of this article has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this article.VVP conceived of the study, and participated in its design and coordination. ARS and EAAS contributed toward the semen analysis. MFO contributed toward the capture and welfare of animals in captivity. All authors, ALCP, TLN, MGCL, and AMLM, contributed to the acquisition and interpretation of data, drafting and revision of this article. All authors have read and approved the final article."}
+{"text": "Background: Faced with the growing burden of cardiovascular disease (CVD) including atherosclerotic in Sub-Saharan Africa (SSA), the development of appropriate prediction tools, based on large cohorts, appears useful for prevention.Objective: The objective of the pilot project TAHES  was to explore the feasibility of a large cohort study focused on CVD and risk factors in Benin.Methods: We implemented a prospective cohort over 2\u00a0years. The sample consisted of all people aged 25\u00a0years or older who had lived for at least the previous 6\u00a0months in the villages of Tanv\u00e8 or D\u00e9kanm\u00e8. At baseline in February 2015, behaviours and medical histories were recorded using a standardized questionnaire adapted from the WHO Steps instrument; screening questionnaires for angina, claudication, congestive heart failure, and stroke were applied; anthropometric measures and fasting capillary blood glucose were taken. All participants were included in the follow-up phase. Surveillance of target CVD and deaths was implemented through a medical and a community network.Results: A total of 1,195 participants were enrolled at baseline; women represented 65.5% and the median age was 39\u00a0years. The high participation rate (91.4%), the quality of baseline data, and the functionality of the events surveillance network over 8\u00a0months indicated good perspective for the feasibility of a large cohort. We recorded a 3.8% prevalence of daily smoking, 3.6% of harmful use of alcohol, 10.7% of obesity, 25.5% of high blood pressure, and 3.5% of diabetes. Prevalence of angina pectoris (2.7%), intermittent claudication (2.0%), congestive heart failure (0.9%), and stroke survival with motor impairment (3\u2030) were also recorded. Ten deaths occurred during the first 8\u00a0months, all within households; a cardiovascular cause was suspected in four cases.Conclusion: These preliminary results support the feasibility of establishing a cohort in Benin. It would require technical and resource support. Cardiovascular diseases (CVD) are the leading cause of death worldwide. In 2012, an estimated 17.5 million people died from CVD, which accounted for 31% of all global deaths; 80% of these deaths occurred in low- and middle-income countries [Sub-Saharan Africa (SSA) has a high burden of both infectious and chronic diseases \u20133. CVD dMajor cardiovascular events can be prevented by early detection and management of people at high risk. Cardiovascular risk scales have been developed from Western cohorts such as the Framingham study  or the SThe TAHES  project aims to develop an appropriate screening tool to identify people at risk of CVD in Benin, applicable to other similar populations in SSA.The objective of the pilot TAHES project was to explore the feasibility of a large cohort study focused on CVD and risk factors in Benin.We planned a cohort focused on CVD in Benin, for at minimum 4 years. Benin is a West African country of around 10 million inhabitants according to the 2013 census . The comThe study concerns adults aged 25\u00a0years and older. A reasoned selection of two neighbourhoods was made.The sample size calculation was founded on the expected cumulated incidence of events in 4 years , Target events are coronary disease, stroke, congestive heart failure, major vascular amputations, and deaths. The definitions of the suspect, likely, and certain cases of each target CVD have been specified.Demographic and lifestyle data, histories of CVD, of hypertension, and of diabetes are collected at baseline. Anthropometrics parameters, ankle brachial index (ABI), and capillary blood glucose are measured. The electrocardiogram recording is planned.The same evaluations are done at two follow-up visits. Events surveillance outside visits is active and continuous through a network of health centres and community sources. The activities are supervised monthly by a physician member of the investigation committee. The scheme of the surveillance is presented in Data are entered in an online secured database by a research assistant using a digital tablet. The database has been created and managed in collaboration with laboratory INSERM UMR 1094 NET . It is created thanks to the Capture System software of the Clinsight company. Capture System is software for management of clinical data using the ORACLE database. The structure and the appearance of the database are unchanged compared with the data collection form.It is accessible online thanks to a personal access (identifier and password). It is stored on the server of CEBIMER . Queries are performed by a data manager and data validated before analysis.The villages of Tanv\u00e8 and D\u00e9kanm\u00e8, located 300\u00a0km north of the economic capital of Benin, Cotonou, were selected for the pilot study. There is a public health centre managed by a nurse. A preliminary census was conducted in the survey area. The number of adults aged 25 and older was estimated to be 1,308.We initiated a prospective, open cohort over 2\u00a0years. All the residents (\u2265 6\u00a0months) in the study area aged 25\u00a0years or older were eligible. Pregnant women and people with major mental deficiency were not included.For the present study, data were collected either in households or in the public health centre of the study area in February 2015. Written informed consent was obtained from each participant. A standardized questionnaire adapted from the World Health Organization (WHO) STEPS instrument  was usedAll participants were included in the follow-up phase. Those people who leave the area are lost from follow-up. New residents, people who reach the age of 25\u00a0years, and women having given birth after a visit are included during the next visit. A network of events surveillance was implemented, involving health centres and community sources. Active and continuous research of events is conducted, based on definitions of suspected and likely cases. A weekly review of care and hospitalization registers at the health centre is done by a trained nurse. In the community, a weekly visit to heads of family, traditional practitioners, and church leaders is conducted by a community agent; a register of death and sickness in the cohort is filled. For every event, a notification form is completed within 24\u201348\u00a0hours and sent to the local investigator (cardiologist). Confirmation and detailed information about notification are provided by the nurse within 7\u00a0days. Verbal autopsy is applied for community death, using the WHO French version 2009  within 2Stroke was defined according to WHO criteria . CongestLow fruit and vegetable intake was defined by the consumption of less than 400\u00a0g of fruits and vegetables per day. Sedentary behaviour was defined by prolonged sitting or sleeping most of the time, \u2265 8\u00a0hours per day. Harmful use of alcohol was defined by consumption of \u2265 3 drinks per day. High blood pressure was defined by systolic blood pressure \u2265 140\u00a0mm Hg or diastolic blood pressure \u2265 90\u00a0mm Hg in one of the two arms, or by currently receiving medication. High blood glucose was defined by capillary whole blood glucose \u2265 6.1\u00a0mmol/L or currently receiving medication.The feasibility criteria are: (1) participation rate at baseline and quality of data, (2) number of events recorded at visits compared with number of events recorded through the continuous surveillance network, (3) percentage of people still followed-up in the cohort after 2 years.The study was approved by Benin\u2019s national ethics committee for health research. Agreements of the local authorities were obtained. Confidentiality is assured at all times during data collection and analysis.The main investigator of the project is a head of the neurology department and director of LEMACEN in Benin. Co-responsible for the project is a chief of the vascular medicine unit of CHU Limoges, a member of INSERM UMR 1094 NET. A local investigator grants 25% of their working time to the project. A PhD student works full-time on the project. Two committees were created. The investigation committee is responsible for study implementation. The scientific committee, comprised of professors in neurology, cardiology, and public health, is responsible for methodology and events validation. Collaborations with two other French teams are planned for the enlarged project.The team of INSERM UMR 1094 NET has already developed common projects in Benin on epilepsy, cognitive disorders, and stroke. They have already conducted a follow-up study on cognitive disorders among an elderly population in the Republic of the Congo.The analysis for this paper were limited to validated data by the time the paper was written. It concerns baseline data and the first 8\u00a0months of follow-up.We used Epi Info 7 to analyze data for this report. Continuous variables were described using the median and the interquartile range. Qualitative variables were described using frequencies and percentages. Chi-square tests or Fisher\u2019s exact test were used to compare proportions between groups. The crude death rate was calculated.There were 1,308 individuals aged \u2265 25\u00a0years eligible in the area and 1,195 accepted to participate (91.4%); 27 refusals were recorded; 70 people were travelling; and 16 were interned in a traditional convent. Women predominated (65.5%). The median age was 39\u00a0years (interquartile range: 30.5\u201355.5). Two thirds of participants (66.4%) had no academic qualifications . Four fiSome criteria indicated good perspective for large cohort feasibility: a high participation rate (91.4%), quality of data collection during the baseline visit, and functionality of the events surveillance network to identify the deaths. The main barrier was the lack of medical information about deaths which occurred in households and illnesses in the cohort.We recorded 3.8% prevalence of daily smoking, 89.5% of low fruit and vegetable intake, and 25.3% of sedentary behaviour ; 8.1% coPrevalence of angina pectoris (2.7%), intermittent claudication (2.0%), congestive heart failure (0.9%), and stroke survivors with motor impairment (3\u2030) were also estimated.Ten deaths, all occurring in households, were notified over 8\u00a0months. The crude death rate was 12.5/1,000 person-years. Causes of death were undetermined in two cases. CVD were suspected in four cases (two sudden deaths and two cases of congestive heart failure). The four other cases were deaths of infectious origin. No new case of non-fatal target CVD was identified; during the period, we identified no cases of hospitalization or care in the local resources for CVD events.The study showed a high participation rate among the source population. People were informed about the follow-up in the study and accepted to participate. They gave their agreement and written consent separately for baseline evaluation and follow-up. The acceptability of follow-up does not seem to be a problem. Previous studies have shown high participation rates at baseline of cohort studies ,38. HighWe noted high levels of high blood pressure (25.5%) and obesity (10.7%), similar to Benin\u2019s national STEPS survey results in 2008 .The crude death rate was 12.5/1,000 person-years. All deaths occurred in households; people who died did not receive medical care at the time of death. They did not attend health facilities at the time of their deaths nor during their sickness. The crude death rate in this study is close to those reported from 22 demographic surveillance sites among people \u2265 15\u00a0years old in Africa and Asia  . In 2010We recorded four cases of deaths probably of cardiovascular origin. This proportion is important and needs to be confirmed by further results. However, this result does not seem to be specific to our study; a high proportion of cardiovascular deaths was previously reported in a South African cohort .CVD causes were based on verbal autopsy and could have been overestimated in our study. Cause-of-death data from SSA are usually not known from death certificates; the high frequency of community deaths was as previously described . This siThe network of events surveillance is functional for recording deaths. The ascertainment of medical conditions is the major challenge. No incident of non-fatal CVD was registered, probably due to default case identification in communities. Strengthening of community networks and health promotion interventions should improve requests for health care and chronic disease surveillance in our study and elsewhere in SSA. Interventions should take into account low levels of instruction, poverty, and social beliefs in rural populations. Besides, health systems should be improved. Visits appear important in this context and are useful for collecting medical data. It appears important to maintain two visits in 4 years as we planned for the TAHES project.Another limitation of the pilot study concerns CVD ascertainment at baseline. CVD are self-reported or based on screening questionnaires. These conditions could have been over- or under-estimated. Not all data planned in the TAHES project were collected in the pilot study taking into account the funding and variables useful for feasibility criteria assessment; electrocardiogram and ankle brachial index were not recorded. We plan to complete these data in April 2017.The assessment of feasibility in this paper is partial. The feasibility of the TAHES project will be completely assessed at the end of the pilot study. However, these preliminary results could allow us to improve the methodology of the TAHES project. They could inform other investigators and guide policies in the study area and other SSA rural areas. They could also support the search for financing for the expanded project. The cost of the pilot study is estimated at 60,000\u20ac for 2 years or approximately 25\u20ac per person per year. The funding of the pilot study has been achieved. The cost of the expanded cohort for 4 years is estimated at approximately 20\u20ac per person per year. The search for funding is in progress.The small sample size in the pilot study is a major limitation. This size is not compatible with precise estimates. It might have not allowed sufficient power to detect any significant differences between groups. Nevertheless the primary aim of this pilot study was to assess the feasibility of a larger cohort. The size of the TAHES cohort will be around 11,000 people; according to our estimation, 120 events will be recorded over 4 years. As attritions and deaths during follow-up might reduce sample size, this cohort will be dynamic.A large cohort study in SSA seems to be technically feasible despite health system weakness and the challenge of ascertaining medical conditions. The study could be expanded after the pilot period.Preliminary results of the TAHES pilot project show good prospects for cardiovascular cohort feasibility in SSA. Strengthening the content of visits and the community network for target events\u2019 surveillance could improve ascertainment of medical conditions in CVD cohorts in SSA rural areas. Establishing a cohort in Benin would require technical and resource support."}
+{"text": "Real-world stressors are complex and multimodal, involving physical, psychological, and social dimensions. However, the brain networks that mediate stress responses to these stimuli need to be further studied. We used c-Fos mapping in mice to characterize brain circuits activated by exposure to a single episode of multimodal stress (MMS), and compared these to circuits activated by electric foot shocks (EFS). We focused on characterizing c-Fos activity in stress-relevant brain regions including the paraventricular nucleus (PVN) of the hypothalamus and the bed nucleus of the stria terminalis (BNST). We also assessed stress-induced activation of CRH-positive neurons in each of these structures. MMS and EFS activated an overlapping network of brain regions with a similar time course. c-Fos expression within the PVN and the BNST peaked 30\u201360\u202fmin after exposure to both MMS and EFS, and returned to baseline levels within 24\u202fh. Quantification of c-Fos expression within BNST subregions revealed that while c-Fos expression peaked in all subregions 30\u201360\u202fmin after MMS and EFS exposure, the neuronal density of c-Fos expression was significantly higher in the dorsomedial and ventral BNST relative to the dorsolateral BNST. Our preliminary assessment indicated that a great majority of MMS or EFS-activated neurons in the PVN were CRH-positive (>87%); in contrast, about 6\u201335% of activated neurons in the BNST were CRH-positive. Our findings indicate that both MMS and EFS are effective at activating stress-relevant brain areas and support the use of MMS as an effective approach for studying multidimensional stress in animal models. The results also reveal that the PVN and BNST are part of a common neural circuit substrate involved in neural processing related to stress. These responses might be highly dependent on the characteristics of stressors . It thusNeural processing of relatively simple stressors such as electric foot shock (EFS) has been intensively studied . HoweverMMS and restraint stress both increase c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and the hippocampus; in contrast, the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) are preferentially activated by MMS, rather than restraint stress . The CeAWhile initial studies suggest that MMS preferentially engages in a specific brain circuit that is not activated by restraint stress , our undIn the current study, we used c-Fos protein immunochemical staining to characterize brain areas activated in mice after a single exposure to MMS, and compared these to brain regions activated by repeated EFS. c-Fos is an intermediate-early gene with activity-dependent protein expression, and has been extensively used to map stimulus-induced neural activation . We foun22.1Wild type C57BL/6J mice acquired from the Jackson Laboratory were used in the experiments. The Cre reporter Ai9 mice  were crossed with CRH-ires-Cre mice  to generate CRH-Cre; Ai9 mice. These mice were used to examine the co-localization of stress-induced c-Fos activation and CRH expression. See the 2.241 C57BL/6J and 8 CRH-Cre; Ai9 mice were assigned to different stress groups: MMS and EFS. Each stress treatment included mice that were randomly divided into groups that differed with regard to the time of sacrifice after stress exposure. The four groups were the unstressed controls, 30\u201360\u00a0min post-stress, 24\u00a0h post-stress, and 1 week post-stress. Control groups for both MMS and EFS did not receive any stress treatment. All four groups  for MMS and EFS, had 4\u20136 wild type mice (N\u00a0=\u00a041). Additionally, the control groups and 30\u201360\u00a0min post-stress groups had two Ai9;CRH-Cre mice to study c-Fos and CRH\u00a0+\u00a0neuron co-labeling per group (N\u00a0=\u00a08). For MMS, the mice were isolated and restrained inside a 50\u202fml closed-ended, perforated conical tube with paper towels filling the residual space. Five tubes with mice were taped to a laboratory shaker in a brightly lit room with loud hip-hop music (at 90\u202fdB) playing for 2\u202fh . InsteadMice were returned to their home cages post-stress exposure. After a 30\u201360\u202fmin wait, 30\u201360\u202fmin post-stress groups were deeply anesthetized and perfused to extract their brains with ventricular blood samples taken. Similarly, 24\u202fh post-stress groups and 1 week post-stress groups were processed at the appropriate time points. All the control mice stayed in their home cages and were perfused together with the 30\u201360\u202fmin post-stress groups.2.3Plasma corticosterone levels were measured using a radioimmunoassay, as described previously . We meas2.4Mice were deeply anaesthetized using isoflurane, blood samples were taken, and transcardially perfused with 5\u202fml of phosphate buffered saline (PBS) followed by 25\u202fml of 4% paraformaldehyde (PFA) in PBS. Brains were extracted and fixed in 4% paraformaldehyde overnight, then stored in 30% sucrose at 4\u202f\u00b0C until cutting.2.5Conventional fluorescent immunohistochemistry was performed on selected brain sections as previously described . Coronal2.62 was calculated as the number of neurons in one region divided by the area size of that region. All statistical analyses were conducted in Sigmaplot13. We combined the control groups of MMS and EFS, because they were not treated differently. We performed a 2-way ANOVA in order to examine stress types  which is the focus of this paper, and time effects. We then performed post-hoc analysis to examine time effects within each stress group. Post hoc comparisons were made using the Student-Newman-Keuls tests. In the case data were not normally distributed, the Kruskal\u2013Wallis analysis of variance and Dunn's Method were used instead. A p value (\u22640.05) was considered statistically significant. All data values are presented as mean\u202f\u00b1\u202fSE.Immunostained slices were scanned under a 10\u00d7 objective of a fluorescent microscope (Olympus BX 61) equipped with a high-sensitive CCD camera and Metamorph software for brain-wide analysis of immuno-labeled tissue. We also imaged labeled neurons in selected sections with a confocal microscope . Most images were obtained using the Metamorph image acquisition software  and analysis were done using Adobe Photoshop (CS4). Slice images were overlaid with corresponding Atlas maps. This enabled us to outline different brain regions. Once the area was measured, Fos-immunopositive neurons were counted manually using a Photoshop counting tool. Consistent with published studies , c-Fos n33.1Mice were subjected to single episodes of either EFS or MMS consisting of concurrent bright light, unpredictable loud noise, jostling (shaker movement) and restraint . To asse3.2To characterize brain circuits engaged by MMS vs. EFS, we used c-Fos, an intermediate-early gene with well-characterized activity-dependent expression , to idenAt 30\u201360\u202fmin after either stress type, strong c-Fos expression was seen in the paraventricular nucleus of the hypothalamus (PVN) and the bed nucleus of the stria terminalis (BNST). In contrast, no labeling was apparent in these areas in control animals. See section At the 30\u201360\u202fmin post-stress time point, there were more c-Fos activation in PAG neurons in the EFS group and the MMS group than stress control animals . In the mediodorsal thalamic nucleus (MD), the data failed the normality test and therefore we ran a Kruskal-Wallis One Way Analysis of Variance on Ranks test with Dunn's Method pairwise comparisons. The controls had less c-Fos expression than EFS, but there was no significant difference between controls and MMS nor MMS vs. EFS . This data is represented in High c-Fos expression was also seen in the prelimbic portion of medial prefrontal cortex (mPFC) and the ventral portion of the lateral septal nucleus (LS) 30\u201360\u202fmin after MMS and EFS. Less expression was seen in the basolateral amygdaloid nucleus (BLA) and there was sparse labeling in the central amygdaloid nucleus (CeA) at 30\u201360\u202fmin after MMS and EFS. Control animals were used to determine baseline c-Fos expression. The qualitative assessments were performed in two cases per condition for these areas and are available in supplementary materials only .3.3The PVN plays a central role in regulating plasma corticosterone levels. To further explore the relationship between stress-induced corticosterone levels and neuronal activation within the PVN, we quantified the time course of c-Fos expression within the PVN of mice that had been exposed to EFS or MMS, and compared this to levels of c-Fos expression in control mice . ExposurThe BNST has been implicated in neural processing related to fear and anxiety states . To furtThe BNST is a heterogeneous structure, and anatomical subregions within this structure have been associated with distinct functional roles . To explWhile the time course of c-Fos expression was similar across BNST subregions, there were pronounced differences in the density of c-Fos expression across anatomical subregions at 30\u201360\u202fmin after MMS exposure , and compared these circuits to those activated following restraint stress. Maras et al. found that both the lateral posterior BNST and the CeA were preferentially activated by MMS vs. restraint stress . While tBoth MMS and EFS caused robust BNST activation in the present study. In contrast, BNST neurons were preferentially activated relative to restraint stress when the prolonged MMS paradigm was used . The mecA large majority of the PVN neurons activated after MMS or EFS were CRH-positive, although it must be noted that this finding is based on preliminary qualitative data based on a few animals. This is consistent with other reports that a variety of stressors lead to robust activation of this population of neurons , and witThere are limitations associated with our use of c-Fos activation to characterize active brain regions after MMS vs. EFS. First among these, the timeline of c-Fos activation may not capture the persistence of neural changes induced by stress induction. In our study, increases in c-Fos expression were observed only at shortly latency (within 30\u201360\u202fmin after stress induction). c-Fos levels returned to control levels of expression within 24\u202fh for both types of stress. After translation, c-Fos protein is degraded relatively quickly, and has a half-life of roughly 1\u202fh . NonetheA second limitation is that c-Fos protein expression is known to vary with neuronal subtype. For instance, neural activity seems to preferentially induce c-Fos expression in excitatory vs. inhibitory neurons within the cerebral cortex and hippocampus . The neuNeural responses underlying behavioral and physiological responses to relatively simple, temporally-delimited stressors , have been well-studied in rodent models. However, the ability of these stressors to capture the complexity of clinically-relevant stressors is not well known. The MMS paradigm used in the present study represents an animal model in which the impact of multiple stressors, with distinct sensory attributes, was compared to foot shock stress. Previous work has shown that long exposure (5\u202fh) to the MMS paradigm results in a pattern of neural activation that is distinct from brain regions activated by restraint stress alone, one of the component stressors comprising the MMS model . However5Both MMS and repeated EFS activated the same brain regions over a similar time course. c-Fos expression peaked 30\u201360\u202fmin after exposure to both MMS and EFS within the PVN and the BNST. At that time point, the neuronal density of c-Fos expression was significantly higher in the dorsomedial and ventral BNST relative to the dorsolateral BNST under both stress conditions. Over 87% of the MMS or EFS-activated neurons in the PVN were CRH-positive whereas 10\u201330% of activated neurons in the BNST were CRH-positive. Our findings support the use of MMS as an effective approach for studying multidimensional stressors in animal models.All authors declare no conflict of interests."}
+{"text": "SLC12A3 (Solute carrier family 12 member 3), which encodes the Na-Cl cotransporter (NCC), and presents with characteristic metabolic abnormalities, including hypokalemia, metabolic alkalosis, hypomagnesemia, and hypocalciuria. In this study, we report a case of a GS pedigree, including analysis of GS-associated gene mutations.Gitelman syndrome (GS) is an autosomal recessive disorder caused by genic mutations of SLC12A3 mutations in a GS pedigree that included a 35-year-old female patient with GS and five family members within three generations. Furthermore, we summarized their clinical manifestations and analyzed laboratory parameters related to GS.We performed next-generation sequencing analysis and Sanger sequencing to explore the SLC12A3 gene identified two novel missense mutations  in the patient with GS. Moreover, we demonstrated that her mother, younger maternal uncle, and cousin were carriers of one mutation (c.1919A\u2009>\u2009G), and her father was the carrier of the other (c.2522A\u2009>\u2009G).The female proband (the patient with GS) presented with intermittent fatigue and transient periods of tetany, along with significant hypokalemia, hypomagnesemia, and hypocalciuria. All other members of the pedigree had normal laboratory results without obvious GS-related symptoms. Genetic analysis of the SLC12A3 and their contribution to GS. Further functional studies are particularly warranted to explore the underlying molecular mechanisms.This is the first report of these two novel pathogenic variants of The online version of this article (10.1186/s12881-018-0527-7) contains supplementary material, which is available to authorized users. SLC12A3 , located on chromosome 16q13, which encodes the thiazide-sensitive Na-Cl cotransporter (NCC) in the renal distal convoluted tubule (DCT).Gitelman et al. first described Gitelman syndrome (GS) as an autosomal recessive disorder presenting with characteristic metabolic abnormalities , includiThe clinical manifestations of GS can be extremely variable, most of which are asymptomatic or mild. It was proposed that GS could be one of the main reasons for low blood pressure in the Japanese population, in which the overall frequency of GS mutations was estimated as 0.0321. Therefore, it was reasonable to expect one subject carrying a heterozygous GS mutation among 15.6 Japanese or 10.3 GS subjects among 10,000 Japanese . HoweverSLC12A3 gene status of a GS pedigree.In the present study, we summarize the clinical manifestations, laboratory examinations, and + supplements since then, during which period her minimum plasma potassium level reached 2.4\u00a0mmol/L. However, her hypokalemia was not properly managed via intravenous K+ supplements, for which reason she was admitted to our hospital.A 35-year-old female with a previous medical history of hypertrophic cardiomyopathy (HCM) for 15\u00a0years was hospitalized to evaluate intermittent fatigue, transient periods of tetany for over 20\u00a0years, and accidentally discovered hypokalemia for 3\u00a0years. For 20\u00a0years, the patient tired easily. In addition, she complained of intermittent self-remitting tetany that occurred after prolonged activity and usually lasted for 5\u201320\u00a0min. Furthermore, she denied other detrimental neuromuscular symptoms . Three years ago, she was found to suffer from hypokalemia (her plasma potassium was 2.7\u00a0mmol/L) after a routine physical examination. She has required treatment with KThe patient took Betaloc for over 5\u00a0years to treat the HCM, but stopped taking the medicine when she prepared for pregnancy. However, she miscarried several times for unknown reasons and currently has no children. The family history indicates that the patient\u2019s parents are not consanguineous. Her sister suffered from sudden death when she was 22\u00a0years old. Her mother and two maternal uncles have been diagnosed with hypertrophic cardiomyopathy for many years.On examination, the patient was conscious. Her blood pressure was 114/76\u00a0mmHg and her heart rate was 110 beats per minute, as measured with a digital electronic sphygmomanometer. Her respiratory rate was 20 breaths per minute and temperature was 36.0 \u00b0C. A 3/6 systolic blowing murmur was heard at the second intercostal space along the left sternal border. Her muscle tone, power, and deep tendon reflexes were normal. The results of other physical examinations were negative.After admission, we monitored her blood pressure twice daily, which showed that the systolic blood pressure was within the range of 94\u2013114\u00a0mmHg, whereas the diastolic blood pressure was within 55\u201370\u00a0mmHg. Electrocardiographic and echocardiographic evaluations were conducted for the patient. She also undertook an adrenal enhanced computed tomography (CT) scan.The patient\u2019s parents, two maternal uncles, and cousin were also evaluated for clinical manifestations and biochemical abnormalities  was collected from each subject. Genomic DNA was isolated from the peripheral blood using a TIANamp Genomic DNA Kit  according to the manufacturer\u2019s instructions.SLC12A3  A-tailing of the 3\u2032 ends to facilitate ligation to sequencing adapters, (ii) ligation of indexed adapters, and (iii) ten PCR cycles to enrich for products with adapters ligated to both ends. Subsequent amplicon sequencing using Illumina miseq PE300 mode was carried out by the Zhejiang Dian diagnostics Co. Ltd.sourceforge.net). Sequence variation annotation was performed using the GATK pipeline (https://software.broadinstitute.org/gatk) and annotated using ANNOVA (annovar.openbioinformatics.org), which comprises gene annotations from multiple sources .From the sequencing reactions, 0.04\u00a0Mb of read data per sample was obtained, and were assessed using stringent bioinformatic filters. The resultant 560\u00d7 average target coverage meant that 100% of the amplicons had at least 30\u00d7 coverage. Sequence reads were aligned to the reference human genome (UCSC hg19) using the BWA software (bio-bwa.SLC12A3 were detected using next generation sequencing (NGS) and subsequently confirmed using Sanger sequencing.Mutation hotspots in Primary laboratory results of the proband are summarized in Table\u00a0The serum potassium, sodium, chloride, calcium, and magnesium levels of the other family members were unremarkable.SLC12A3 (c.1919A\u2009>\u2009G and c.2522A\u2009>\u2009G) in the proband, which resulted in substitution of Asparagine (N) 640 by Serine (S) and of Aspartic acid (D) 841 by Glycine (G), respectively  to characterize the effect of the novel variants (c.1919A\u2009>\u2009G and c.2522A\u2009>\u2009G) on the protein structure of NCC, which demonstrated that the alterations resulting from the mutations altered the protein structure.We used the SWISS-MODEL workspace  in 1996 and revealed the molecular basis of GS, which is genetically distinct from Bartter syndrome.Gitelman et al.  first de+ excretion [SLC12A3 result in NCC dysfunction, producing a clinical disorder that presents with activation of the renin-angiotensin-aldosterone system together with secondary potassium-wasting and metabolic alkalosis.NCC is mainly expressed in the epithelial cells of the distal tubule, which can be blocked by thiazide diuretics to enhance Naxcretion . The varIn addition to hypokalemia, metabolic alkalosis, and increased renin activity, the patient in our case also presented with hypomagnesemia and hypocalciuria, which are two indispensable criteria for diagnosing GS. Intriguingly, the mechanisms responsible for hypomagnesemia and hypocalciuria in patients with GS remain elusive. The serum magnesium balance is maintained via both intestinal absorption and renal excretion. Distal reabsorption of magnesium mainly occurs in the renal DCT via several transcellular transporters, among which the transient receptor potential channel melastatin subtype 6 (TRPM6), located at the luminal membrane, is responsible for magnesium entry from the tubular lumen into the DCT cells . NijenhuS707X/S707X mice exhibited increased expression levels of TRPV5 and TRPV6 in the DCT, Yang et al. [Meanwhile, the mechanisms responsible for hypocalciuria remain controversial. The majority of calcium reabsorption occurs in the proximal convoluted tubule (PCT) and the thick ascending limb of the loop of Henle through passive diffusion, in which process, approximately 65 and 20% of calcium is reabsorbed in these sites, respectively. About 10\u201315% of filtered calcium reabsorption occurs through active transport in the DCT, during which period the gate keepers are the transient receptor potential cation channel subfamily V member 5 (TRPV5) and member 6 (TRPV6) . Nijenhug et al.  proposedIn our case, the patient also suffered from metabolic complications, such as hyperuricemia and impaired glucose tolerance. Thiazide diuretics can elevate serum uric acid (UA) by up to 35% . There aThe proband, her mother, and two uncles were diagnosed as HCM for many years, which is an autosomal dominant inheritance disease caused by mutations in genes encoding sarcomeric proteins . ElectroSLC12A3 mutations [SLC12A3 mutations together with male gender [The clinical manifestations in patients with GS are extremely variable, not only in patients carrying different utations , but alsutations , and eveutations , 27. Cure gender .SLC12A3 gene have been discovered in patients with GS , including missense mutations, shear mutations, nonsense mutations, and frame shift mutations, among which missense mutations are the most commonly described. Moreover, compound heterozygous mutations are more common than homozygous mutations [To date, 488 mutations of the utations .SLC12A3 mutations in 448 patients suspected of having GS using direct sequencing of genomic DNA. They identified two mutations in 315 patients (70%); one mutation in 81 patients (18%); and no mutation in 52 patients (12%). Among patients suspected of having GS, 18\u201340% carried only one mutant allele; therefore, researchers further screened for large rearrangements, which are often missed by direct sequencing. The results demonstrated that large rearrangements accounted for approximately 6% of the mutations in GS [Researchers screened for ns in GS .To the best of our knowledge, we identified the compound heterozygous variants  for the first time. The patient\u2019s mother, younger uncle, and cousin were the carriers of one variant (c.1919A\u2009>\u2009G), and her father was the carrier of the other (c.2522A\u2009>\u2009G). However, the patient was the only member of the pedigree presenting with apparent clinical manifestations and suffering from GS. Such cases have been reported before , 31. In The modeled structure predicted by the Swiss-Model Workspace suggested that c.1919A\u2009>\u2009G alone caused three visible differences in the protein structure and that c.2522A\u2009>\u2009G alone caused one more difference. Furthermore, coexistence of c.1919A\u2009>\u2009G and c.2522A\u2009>\u2009G resulted in three more differences compared with c.2522A\u2009>\u2009G alone. Thus, we hypothesized that the presence of more mutations would cause more structural alterations leading to protein dysfunction. However, further functional analyses are needed to establish the relationship between the genotype and phenotype.How do these mutations lead to protein dysfunction? Previous functional research proposed at least five different underlying mechanisms involving the reduced or abolished transport activity, which could be summarized as follows: (1) impaired protein synthesis; (2) defective protein processing; (3) defective protein insertion; (4) protein dysfunction with impaired intrinsic activity; and (5) accelerating the degradation of protein , 33.SLC12A3 mutations because 14 of the 20 mutations identified in their study were located in this region. The C-terminal end contains three acknowledged protein kinase phosphorylation sites. Given that these sites are indispensable to maintaining the protein\u2019s function, and regulate many ion transport systems, it is reasonable to suppose that mutations in this domain might alter the protein configuration to interfere with phosphorylation, resulting in defective NCC activity.NCC is a 1030-amino-acid integral membrane protein with 12 transmembrane domains and intercellular N- and C-terminal domains  located in the C-terminal domain of the NCC protein were identified in the proband. One variant (N640S) was inherited from her mother and the other (D841G) was inherited from her father. In view of their locations, we presumed that these two novel variants would cause impairment or loss of NCC function. Further functional studies are warranted to investigate the molecular basis of the defective NCC function caused by these two possible pathogenic variants.Additional file 1: Table S1.Clinical and biochemical characteristics of all five family members. Clinical and biochemical characteristics of all five family members are listed in Table S1. (DOCX 14 kb)Additional file 2: Table S2.SLC12A3 gene and annealing temperature of each pair. Thirty-three primer pairs designed to amplify the coding sequence of the SLC12A3 gene, and annealing temperature of each pair, are provided in Table S2. (DOCX 14 kb)Primer sequences of the"}
+{"text": "Synapsis ritsemae. We found intraspecific differences in morphological traits across different land use types. Phenotypic plasticity was found for traits associated with dispersal (wing aspect ratio and wing loading) and reproductive capacity (abdomen size). The ability to predict behavioral functional traits from morphology is useful where the behavior of individuals cannot be directly observed, especially in tropical environments where the ecology of many species is poorly understood. In addition, we provide evidence that land use change can cause phenotypic plasticity in tropical dung beetle species. Our results reinforce recent calls for intraspecific variation in traits to receive more attention within community ecology.Functional traits and functional diversity measures are increasingly being used to examine land use effects on biodiversity and community assembly rules. Morphological traits are often used directly as functional traits. However, behavioral characteristics are more difficult to measure. Establishing methods to derive behavioral traits from morphological measurements is necessary to facilitate their inclusion in functional diversity analyses. We collected morphometric data from over 1,700 individuals of 12 species of dung beetle to establish whether morphological measurements can be used as predictors of behavioral traits. We also compared morphology among individuals collected from different land uses  to identify whether intraspecific differences in morphology vary among land use types. We show that leg and eye measurements can be used to predict dung beetle nesting behavior and period of activity and we used this information to confirm the previously unresolved nesting behavior for  Dung beetles can be separated into distinct functional groups based on body size, period of activity , and nesting behavior. They can nest within a dung pat (endocoprids or dwellers), beneath the dung (paracoprids or tunnellers), or by rolling a dung ball horizontally away from the source (telocoprids or rollers) : old\u2010growth forest (OG) which had never been logged; logged forest (LF) which had experienced 2\u20133 periods of timber extraction, and oil palm plantations (OP) in which palms were planted between 2000 and 2006. All sampling points were located within the Stability of Altered Forest Ecosystems (S.A.F.E.) project, encompassing the old\u2010growth forest at Maliau Basin,  were sampled in Sabah, Malaysian Borneo during February and March 2011. We sampled nine sampling points at three sites in each of three land uses , Boucomont (1914), the works on Bornean Scarabaeinae by Ochi and Kon . Species names, functional groups  as its nesting behavior is unresolved. Synapsis ritsemae is the only member of its genus to occur in Borneo. This genus has traditionally been classified as a nocturnal tunneller based on its taxonomic position within the Coprini  to test whether morphology differed between functional groups. Data on hind leg size, hind leg robustness, eye roundness, and eye size were used as the response variables, with functional group  as the predictor. Site was used as a grouping variable to account for spatial autocorrelation. To visualize the differences between functional groups we used a principal component analysis (PCA).3.2p\u00a0>\u00a00.05. A Gaussian error distribution was appropriate for all response variables. We excluded data on minor males, as there were insufficient measurements for all species across all land uses to estimate the Land use:Sex interaction with sex as a three\u2010level factor . We selected focal species that occurred in more than one land use type. However, because dung beetles show strong affinities to particular land uses, many species were not found in all three land uses. Therefore, for each morphological trait we ran separate models for the species occurring in:We tested whether dung beetle morphological traits varied across land uses with general linear mixed models (GLMMs). We specified land use , species, sex , and the two\u2010way interactions Land use:Species and Land use:Sex as fixed effects, and set site as a random factor. Model selection was based on p values, starting with the full model and dropping terms for which model comparison using chi\u2010squared tests gave n\u00a0=\u00a01,315 individuals measured from 11 species across six sites: Catharsius dayacus, Copris sinicus, Microcopris doriae, Onthophagus obscurior, Onthophagus rugicollis, Onthophagus vulpes, Paragymnopleurus maurus, Paragymnopleurus sparsus, Paragymnopleurus striatus, Proagaderus watanabei, and Sisyphus thoracicus).Set A) Old\u2010growth and logged forest .Set B) Logged forest and oil palm  with a quasibinomial error structure to account for overdispersion. Land use, species, and the two\u2010way interaction were fixed effects. As above, we separated data into comparisons of logged forest and old\u2010growth forest (39 comparisons of seven species across six sites) and logged forest versus oil palm (21 comparisons of four species across six sites), and used model selection as described above.All analyses were run using the R software . Differences between functional groups were visualized using a principal components analysis  of different functional groups  showed significantly higher wing loading in old\u2010growth relative to logged forest sites, and one species (Proagaderus watanabei) showed lower wing loading , Land Use alone was significant Table\u00a0. Wing asOnthophagus rugicollis, that had larger hind leg size and abdomen size in oil palm compared to logged forest. The other species showed no response in relation to land use . There were no significant Land use:Sex interactions; females and major males showed similar differences between logged forest and oil palm. Across all species found in logged forest and oil palm, morphological measurements differed significantly between males and females apart from hind leg size. In each case, males were larger apart from wing loading, which was larger in females.Intraspecific trait differences between oil palm and logged forest only occurred in two of the six morphological traits measured: hind leg size and abdomen size Table\u00a0. For botProagaderus watanabei, Onthophagus obscurior, and Onthophagus rugicollis). By comparing Figures\u00a0There were three species that were found in all three land uses , nor in species found in logged forest compared to oil palm .There were no statistically significant differences between the proportion of major and minor males in species found in logged forest compared to old\u2010growth forest  and reproductive capacity (abdomen size).5.1Dung beetle functional groups are most often classified based on direct observation of behavior , or to move beyond discrete classifications of behavior to metrics on a continuous scale (Fountain\u2010Jones The approach of classifying function through morphology could be extended to distinguish more precise, refined behavioral functional group classifications for dung beetle species (such as 5.2Changes in dung beetle functional diversity across different land uses have been previously recorded (Andresen, We found evidence of intraspecific differences between old\u2010growth and logged forests for several of the traits measured. Wing aspect ratio was the only trait that did not show intraspecific variation; where relatively shorter, wider wings were found for individuals of all species in logged forest in comparison with old\u2010growth. Abdomen size and wing loading were found to be on average larger across all species in old\u2010growth in comparison with logged forest. Relatively wider and shorter but smaller wings and smaller abdomens in logged forest could be indicative of a trade\u2010off between dispersal traits (Gibb et\u00a0al., Larger bodied dung beetle species are thought to be more vulnerable to the effects of habitat disturbance, showing both reduced abundance and species richness in degraded forest (Larsen, Williams, & Kremen, Hind leg size and eye size varied between species, and land use comparisons. This suggests that changes in resource availability, temperature exposure, and habitat structure do not have the same effects on all species or all traits. For example, eye size was found to be larger for some species in logged forest in comparison with old\u2010growth forest, and smaller for others. Thus, any possible explanation for changes in trait values affecting visibility and eyesight, such as changes in light levels through canopy structure, cannot be acting on all species in the same way. Similarly, other processes, such as competition between species, and the trade\u2010offs of size in any morphological dimension, appear to be acting differently on different species.6We have demonstrated that behavioral traits can be resolved from morphology in dung beetles, using seven morphological traits to successfully confirm or identify five behavioral traits, associated with nesting behavior, dispersal, and activity period in 12 dung beetle species. This indicates the potential to further the use of morphological characteristics in defining functional traits in dung beetles and other groups, and their use to support and supplement the classification of behavioral traits. Our findings also support recent calls for increased reporting and use of intraspecific variation in studies of morphological and functional traits (Bolnick et\u00a0al., None declared.CG, DJM, and EMS conceived the study. EMS collected the samples , EHR carried out morphometric data collection and DJM assisted in species identification. CG, EHR, and EMS carried out data analysis. EHR lead the writing of the manuscript with input from all authors.https://doi.org/10.5061/dryad.1484dn3.Dung beetle morphometric data can be accessed the Dryad Digital Repository: \u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "To enhance enrollment into randomized clinical trials (RCTs), we proposed electronic health record-based clinical decision support for patient\u2013clinician shared decision-making about care and RCT enrollment, based on \u201cmathematical equipoise.\u201dAs an example, we created the Knee Osteoarthritis Mathematical Equipoise Tool (KOMET) to determine the presence of patient-specific equipoise between treatments for the choice between total knee replacement (TKR) and nonsurgical treatment of advanced knee osteoarthritis.With input from patients and clinicians about important pain and physical function treatment outcomes, we created a database from non-RCT sources of knee osteoarthritis outcomes. We then developed multivariable linear regression models that predict 1-year individual-patient knee pain and physical function outcomes for TKR and for nonsurgical treatment. These predictions allowed detecting mathematical equipoise between these two options for patients eligible for TKR. Decision support software was developed to graphically illustrate, for a given patient, the degree of overlap of pain and functional outcomes between the treatments and was pilot tested for usability, responsiveness, and as support for shared decision-making.r2 value of 0.32, and the model for physical functioning included six patient-specific variables, and an r2 of 0.34. These models were incorporated into prototype KOMET decision support software and pilot tested in clinics, and were generally well received.The KOMET predictive regression model for knee pain had four patient-specific variables, and an Use of predictive models and mathematical equipoise may help discern patient-specific equipoise to support shared decision-making for selecting between alternative treatments and considering enrollment into an RCT. The ethical and scientific basis for randomly assigning treatments in a randomized clinical trial (RCT) is the presence of clinical equipoise, the absence of a clearly superior treatment. However, this is typically not an individual patient-centered determination, but rather based on inference from groups defined by pivotal studies\u2019 inclusion and exclusion criteria.An alternative would be to compare patient-specific predictions of treatment outcomes, if available. If such predictions are generated by mathematical models that account for individual patient characteristics, then the potential outcomes can be compared, looking for \u201cmathematical equipoise\u201d . Therebynot appropriate for random treatment assignment, for whom this decision support could enhance care by indicating the potentially superior treatment for a given patient. This could help make transparent the basis for selection for an RCT in real time and enhance fully informed consent in the midst of clinical care. It could facilitate patient\u2013clinician shared decision-making, both in care and in the decision to participate in an RCT.If there is the capacity to identify patient-specific equipoise embedded in electronic health records (EHRs), it could serve as a practical way in routine clinical care to detect potentially eligible patients for RCT enrollment. It also could identify those We previously created and tested predictive instrument decision aids based on multivariable logistic regression models that provide 0%\u2013% predictions of medical diagnoses and treatment outcomes \u20135. TheseDecision support for more complex and longer term decisions benefits patients when they improve knowledge, accuracy of risk perceptions, and concordance with patient values . They alShared patient\u2013clinician decision-making is central to choosing between medical treatments and surgical total knee replacement (TKR) for long-standing knee osteoarthritis. The shared decision process involves the clinician and patient, and others as desired, sharing information about treatment options and their risks and discussing patient preferences and values . PatientSymptomatic knee osteoarthritis, estimated as affecting 17%\u201334% US adults , is the In this context, we sought to create the Knee Osteoarthritis Mathematical Equipoise Tool (KOMET) to be embedded in EHRs as decision support for shared clinical decision-making. KOMET was intended to identify patients for whom, based on their specific characteristics, there is no sufficient evidence to favor medical or surgical treatment. This is a circumstance in which shared patient\u2013clinician decision-making is important \u2013 when patients\u2019 personal preferences and objectives may dominate what might appear as a \u201ctoss-up\u201d decision . This alTo create the mathematical models for our prior predictive instruments, we used data from RCTs. Using data from treatments that were randomly assigned avoided the treatment effects being biased by the selection of their use among patients. Thereby, the multivariable regression models more accurately reflect the effects of treatments when used in comparable patients. However, there are many conditions and treatments for which RCT data are not available. Indeed, for the very circumstances that would call for an RCT, for which predictive models and mathematical equipoise might be wanted to assist participant selection, there often will be little RCT data on which such models could be based. Therefore, to create predictive models for this purpose, we must use data from other sources, including observational studies, registries, EHR-based data warehouses, and patient-acquired data. If these non-RCT sources could be used for creating predictive models, there would be vast opportunities for the mathematical equipoise approach to facilitate the conduct of clinical effectiveness RCTs. However, there are protean challenges and limitations.This project sought to create KOMET as an example of the use of mathematical equipoise for determining patient-specific equipoise. As an example of this approach, we used patient-level data from existing non-RCT sources to build predictive models of treatment outcomes to determine the presence or absence of patient-specific equipoise, to inform decision-making. Demonstrating the ability to do this could support wider applicability of the mathematical equipoise method.To develop the KOMET\u2019s predictive models, we created a consolidated database with treatment outcomes of knee osteoarthritis from clinical studies and patient registries. Model variables and predicted outcomes were selected based on input from stakeholders, availability of data, and variables\u2019 contributions to models\u2019 predictive performance. When completed, the models were incorporated into prototype decision support software and tested with clinicians and patients. Throughout, we involved stakeholders with an interest in the outcomes of the project including patients and their families, advocacy group representatives, clinicians, and researchers .The Multicenter Osteoarthritis Study (MOST), an NIH-sponsored longitudinal, prospective, observational study of knee osteoarthritis in adults with osteoarthritis or at increased risk of developing osteoarthritis [http://most.ucsf.edu [rthritis . The datucsf.edu .The Osteoarthritis Initiative (OAI), an NIH-sponsored multicenter, longitudinal, prospective observational study of osteoarthritis intended as a public domain research resource that includes 4,796 men and women aged 45\u201379 who have, or are at high risk for developing, symptomatic knee osteoarthritis. Data were obtained from http://oai.ucsf.edu [ucsf.edu .The New England Baptist Hospital (NEBH) Orthopedic Surgery Registry of 2,462 patients who underwent TKR since 2011 [nce 2011 . AssessmThe Tufts Medical Center (TMC) Orthopedic Surgery Registry of 535 patients who had TKR surgery since 2007 [nce 2007 . AssessmTo create the modeling database, we used the following databases, which are described in more detail elsewhere :The MultThe Western Ontario and McMaster Universities Arthritis Index (WOMAC) [ (WOMAC) , developThe SF-12\u00ae Health Survey a multipurpose \u201cshort-form\u201d generic measure of health status [h status , 27. It h status .The models created on these databases predicted clinical outcomes expressed as scales; for pain, the WOMAC Index  and for The database for creating KOMET models included two types of registries. MOST and OAI had data collected on knee osteoarthritis at fixed intervals per their protocols, including those who underwent TKR and were continued to be followed. The other two registries, NEBH and TMC, were from hospitals that collected baseline and follow-up data only on their patients who had TKR.For KOMET, our target patients were those with knee osteoarthritis who had reached the clinical state at which they would be making a decision of whether or not to have TKR. Lacking a cohort of such patients randomized to medical or surgical options, we used data from patients who had TKR, and matched them to patients (knees) who did not have TKR, but otherwise had similar characteristics. To do this, we created a database in which the \u201cknee\u201d was used as the unit of analysis, and matching was done based on characteristics of the knee and the patient. Thereby, we created a study sample of pairs of similar patients who could be considering this therapeutic choice.closest previous visit as the \u201cbaseline\u201d visit for that TKR including demographics, knee characteristics, comorbidities, mental and physical function, and other clinical features. To find non-TKR \u201ccontrol knees,\u201d we created a subdatabase of all knee visits of all patients, excluding any that occurred after a TKR. We then used a \u201cgreedy\u201d matching computer algorithm [For the MOST and OAI registries, we identified all knees that underwent TKR and then used data collected at the lgorithm  to selecThe variables used for matching differed among the databases, based on data availability. As a guide to determine variables to use for matching, we used input from the research team, clinicians, stakeholders, and the literature . For matAnalyses were done using SAS for Windows, version 9.4 TS Level 1M2, Copyright \u00a9 2002-2012 by SAS Institute, Cary, NC and SAS Enterprise Guide, version 7.13 HF3 . More details of modeling can be found in a separate article [We developed a multivariable linear regression model for the 1-year knee pain outcome based on the WOMAC score, or when a database lacked WOMAC items, using an estimated WOMAC score. Our approach was to develop the model using a set of matched TKR to non-TKR knees from the OAI database, then to validate/test it on a set of matched TKR and non-TKR knees from the MOST database, and then update it by pooling the OAI and MOST datasets and building a new model, starting with variables used in the model developed in the OAI data and tested on the MOST data. We also rederived models, using a similar variable selection process, but with a more limited set of candidate predictor variables, in order to include NEBH and Tufts datasets together with OAI and MOST. This entire process was repeated for the 1-year functional outcome . To create models that could provide predicted estimates of 1-year knee pain and 1-year function, with and without TKR, for any patient based on their characteristics, all models included a treatment indicator (interaction) variable. Covariates and interactions of treatment with covariates were explored in the different phases of the modeling process. No adjustment for matching was done in the linear regression during modeling. This was because the purpose of matching was to create a reasonably balanced study sample of patients who either had TKR or did not have TKR with comparable characteristics, with covariates in the models accounting for remaining imbalances between the groups .The goal of software development and usability testing was to translate the results of the predictive models into easily understood, patient-specific reports that could be produced in the course of clinical care for shared treatment decision-making and, if appropriate, enrollment into an RCT.Decision Support Software Development. There were two KOMET software development tasks: one for the analytics and another for the user interface. Analytics development included implementing the predictive models as reusable, multiplatform software components to generate the current and 1-year-predicted pain and function outcomes for nonsurgical and surgical treatments. The analytics software also calculated the respective 95% confidence intervals around each prediction as the basis for considering the degree of overlap that would suggest near equivalence or patient-specific equipoise. User interface development included creating a web browser-based questionnaire interface to collect patient demographics, items for computing the WOMAC pain score, the SF-12 physical functioning scale, and comorbidities. Together, the analytics and user interface components included methods for data retention and presentation of the predicted outcome results.Then, the predictive models then were incorporated into the web-based decision support application for iterative user testing. Usability testing included a \u201cthink-aloud\u201d protocol and a usability-testing script administered by research staff. The IRB determined that the project was exempt from IRB review.Mathematical equipoise was defined for KOMET as when the pain and functioning outcome predictions with nonsurgical care and TKR are close, i.e., within, or overlapping, each other\u2019s \u201cuncertainty circle.\u201d In Fig. The final database included 1,452 knees (726 with TKR and 726 without) of 1,322 patients. Ninety-one percent of patients  had a single knee included in the database, 8% (106) had two knees used or a single knee used two times, and 1% (12) had their knees used three times. OAI TKR knees were matched to control knees from OAI, and MOST TKR knees were matched to controls from MOST. Because NEBH and TMC included only TKR knees, their controls were drawn from non-TKR knees from OAI and MOST. In the final matched database, the relative contributions of TKR knees were OAI, 252; MOST, 154; NEBH, 248; and TMC, 72. For the control knees, contributions were OAI, 472, and MOST, 254.2. On the 0\u2013100 pain scale (100 indicating extreme pain), the mean baseline knee pain was significantly higher in the TKR than non-TKR group , despite efforts to match on this variable (categorized). Baseline characteristics of interest to clinicians and stakeholders that were considered for the modeling process were comparable between TKR and non-TKR knees, and also for the variables used in the final models, using imputed data. Irrespective of significance, all variables listed in Table Characteristics of the study sample are summarized in Table P = 0.008). Overall, at follow-up, there was less knee pain and better physical function in the TKR than the non-TKR groups. Distributions of variables used for the matching process confirmed that in each database, characteristics used for matching were well balanced between the TKR and non-TKR knees.Comparisons of mean SF-12 scores between TKR and non-TKR groups showed better physical and mental function in the non-TKR groups than TKR groups, with the difference being significant for physical function  and the SF-12 physical functioning component score. Based on extensive discussions with patient and clinician stakeholders, 1 year was chosen as the target follow-up time to have a time point beyond recovery from surgery (estimated as up to 9 months). Stakeholders felt benefits of surgery were stable beyond that time point, and so to address data inconsistencies and gaps, data from up to 5 years past baseline were allowed for use when no closer follow-ups were available.The final predictive models for pain and physical functioning are presented in Table We used these models to predict 1-year knee pain and physical functioning for the treatment each subject underwent (TKR or non-TKR), and also for the counterfactual  treatment. These data allowed us to predict the difference in pain and function outcomes for each patient for TKR and non-TKR. The distribution of predicted differences is in Fig. http://medicalequipoise.com/tkrclinician) and one for patients . Both applications are composed of an analytics software library that also could be embedded into an EHR system.The KOMET development process resulted in the creation of one web-based application for clinicians . The impact of CER is based on evidence generation, which then leads to evidence synthesis, interpretation, application, dissemination, implementation in widespread practice, and then feedback for the generation of new evidence. Ideally, this entire chain rests upon having unbiased and generalizable RCT evidence. An EHR-based method for patient-centered enrollment into RCTs should allow more targeted comparative effectiveness trials in more diverse clinical sites and more representative participants, thereby applicable to more patients and more care settings. Moreover, more complete EHR-based identification of potential participants could reduce clinical trial duration, bringing results to the public sooner and reducing costs.Aside from these potential benefits, we hope that this method might facilitate overall care by promoting conversations between clinicians and patients based on evidence specific to their care. This also could facilitate clinicians\u2019 and the public\u2019s understanding of, and participation in, clinical trials.Although not ready for widespread implementation, in its content, user interface, and connectability to EHRs, KOMET did function as intended. We believe it warrants further development for implementation in clinical settings, even while KOMET has limitations related to the available data, modeling methods, analytic methods, and the prototype software.An important limitation of our approach is that the models were created on potentially biased data. We sought data from studies that had both surgical and medical treatment of knee osteoarthritis, and two of our studies had that, but two other registries included only one treatment (surgery). Both types of sources provide challenges for creating comparable patients who underwent the two treatments, as is needed to make accurate models of the two treatments. However, for mathematical equipoise to serve its intended purpose of facilitating RCTs of treatments for which none have yet been done, its models will need to be made on non-RCT data, as was the case in this project. We undertook many checks to accurately represent effects despite the likely biased samples. KOMET\u2019s models performed well despite this challenge, but for wider use of this approach, additional sources of data and analytic methods should be developed.The modeling methods also have limitations. Multivariable regression methods we used have advantages over some more computer-intensive methods, including their clearly interpretable variable coefficients and resistance to overfitting, compared with some computer-driven methods . HoweverIn addition to the modeling methods, the variables we used have limitations. Based on the collection of important variables in the available databases, published clinical evidence, and stakeholder input, we believe we used very credible variables to represent independent and dependent (treatment outcome) variables. However, there is a specific limitation in the functional outcome we predicted, which reflected our intent to capture a holistic physical function of the patient, based on the SF-12 functional scale. In looking at the results of the KOMET\u2019s predictions, we noticed that pain is often very substantially changed by surgery, but function tends to have a relatively modest improvement. In discussing this with patients, it appears possible that we might have better captured their meaningful knee functional improvement if we had used a more knee-specific function rather than overall physical functioning. Meetings with stakeholders suggest that both overall and knee-related function are important, and future work should develop predictions of the more specific knee-related function would be useful to both patient and clinical stakeholders , 34. TheWe believe the basis for patient-specific equipoise, mathematical equipoise will benefit from further development, including a generalizable method by which the overlap of two patient-specific predictions can be designated as sufficient to constitute equipoise. In addition, as we learned from our clinical testing, ways to effectively convey the meaning of patient-specific equipoise deserve development such that is clear to patients and clinicians.The prototype KOMET software has limitations. The creation of full-featured, user-friendly, robust software was beyond the scope of this project. Our prototype needs significant further development before it could be used in routine care. Nonetheless, we believe that it is quite attractive and functional, and in the context of its intended role in this project, a successful product of this project. More research is needed for this type of decision support, which should be full-featured, user-friendly, interoperable, and robust, and will be attractive to help identify patients for whom enrollment in an RCT might be appropriate.The limitations listed above all suggest areas for future research. Approaches must be developed that lessen the biases inherent in clinical registry data. Just having more data, such from EHR data warehouses and other sources, will not eliminate biases. Finding ways to mitigate the biases, using selection and sampling methods and other approaches, will be extremely important for work on mathematical equipoise, and for many other efforts to harvest clinically important insights from clinical data.Modeling clinical outcomes based on data is evolving rapidly, and increasingly sophisticated computer-based methods, such as artificial intelligence and machine learning, are being applied to analysis of clinical data . AlthougThis project demonstrated the use of predictive instruments and mathematical equipoise as a method for detecting patient-specific equipoise between alternative treatments. Embedded in EHRs, this approach should help identify patients for whom one or the other treatment seems likely to yield better outcomes based on their specific characteristics, and also patients for whom there is insufficient evidence to favor one treatment. This can be part of a shared decision-making process that incorporates the patient\u2019s preferences and priorities, and it also, as clinical equipoise, can support enrollment into an RCT. This approach will require further development, and will benefit from testing in clinical practice and for facilitating comparative effectiveness trials."}
+{"text": "The explosive behaviour of concrete in fire is observed in rapidly heated concrete. The main factors controlling the occurrence of spalling are related to the material\u2019s low porosity and high density as well as the limited ability to transport gases and liquids. Thus, for high-strength, ultrahigh-strength, and reactive powder concrete, the risk of spalling is much higher than for normal-strength concrete. The paper presents the discussion on the leading hypothesis concerning the occurrence of concrete spalling. Moreover, the methods for spalling prevention, such as polypropylene fibre application, which has been found to be an effective technological solution for preventing the occurrence of spalling, are presented. Various tests and testing protocols are used to screen concrete mixes propensity toward spalling and to evaluate the polypropylene fibres\u2019 effectiveness in spalling risk mitigation. The most effective testing methods were selected and their advantages were presented in the paper. The review was based mainly on the authors\u2019 experiences regarding high performance concrete, reactive powder concrete testing, and observations on the effect of polypropylene fibres on material behaviour at high temperature. During a fire, it is possible that concrete, which is an incombustible material , will beSpalling may take the form of a very slow detachment of the surface layer of the material, as well as of pieces of material, which break away in a rapid, explosive manner from the element . Differe1 and t2 indicated in the figure relate to different points in time showing the progressive character spalling type. In this subdivision, the corner (A), the aggregate (B), the surface (C), and the explosive spalling (D) were represented.Observations of fire damage to real structures and laboratory fire tests have made it possible to distinguish five different types of spalling, which were recently summarised by the authors . The folT between the heated layer, which expands, and colder concrete layers. This is usually not dangerous for the load-bearing capacity of structures, since it generally does not result in the loss of a significant part of the cross-section and does not become exposed in reinforcing steel. Explosive spalling (C), which is accompanied by characteristic explosion sounds, constitutes a violent chipping off of concrete pieces, with large surface areas and spalling depth reaching up to around a dozen centimetres for a single piece of spalled concrete. Explosive spalling of concrete results in reinforcing steel becoming exposed and often results in the complete destruction of the concrete layer. Corner spalling (D) is observed in elements in which two orthogonal surfaces, which form a corner , are heated. In a typical fire, corner spalling usually takes place for about 30 min after the fire has started. Corners are heated more quickly, which results in higher thermal gradients at such points, and, consequently, corners are damaged more quickly as well. The peeling of concrete after a fire (E) characterises heated concrete, where dolomite and calcareous aggregates occur during the cooling of the structural element. This may take place a few hours or even days after the fire has ended. The peeling of damaged concrete layers after the fire is the result of an inversed temperature gradient during cooling. Nevertheless, the main cause of post-fire concrete peeling (concrete sloughing off) is the hydration of calcium oxide, formed during decomposition of portlandite, calcareous, and limestone aggregate, which is accompanied by the expansion of the reaction product [The occurrence of different types of spalling is accompanied by characteristic sounds\u2014popping for the aggregate and surface spalling  and a loud bang for the explosive one (C). Some regularity in the occurrence of spalling, according to the type of spalling, was observed. During the first minutes of a fire or fire test, spalling is most likely to take place in the aggregate and surface. Then, after a few minutes, a loud bang of explosive behaviour appears, and afterwards, corner spalling takes place. Usually, aggregate spalling (A) does not affect the load-bearing capacity of a structure and does not lead to a significant cross-section loss. It is believed that the mechanism behind aggregate spalling is similar to a flaming stone procedure, which results from quartz transformation. Surface spalling (B) affects the thin and flat fragments of the near-surface layers of concrete elements, such as walls and ceilings, which become detached from the surface as a result of the tensile stress \u03c3 product .Several hypotheses concerning the occurrence of spalling have been proposed ,4. CurreDuring a fire, a temperature increase leads to concrete drying. The thermal gradient is also responsible for water migration. As the temperature increases, the water moves toward colder zones due to the thermo-capillary effect. Moreover, the water vapour that is produced during heating moves through an open network of concrete pores. The gas transport continues, until the thermodynamic conditions are satisfied for vapour condensation. Under this condition, a layer of material, saturated with water, is formed, which is called a moisture clog zone. The theory of a moisture clog was confirmed experimentally, in Reference , from anAs a consequence, the water-clogged zone is more prone to forming in high-strength concretes (HSC) and ultra-high-performance concretes (UHPC), in which the permeability is 0.52E-17 . The redIt needs to be highlighted that the external load has a critical effect on the spalling behaviour of a concrete element. It was observed that the external compressive load or pre-stress enhances the risk of spalling ,20,21. A3), is placed horizontally and is directly exposed to an elevated temperature on the upper surface using radiant heaters, as shown in The gas pore pressures can be measured while heating using pressure gages embedded in the concrete element during casting. Kalifa et al. successfully measured the gas pore pressure and temperature development during heating , reporti3 of fibrillated PP fibres. The PTM test also serves as a calibration test for all thermo-hydro-mechanical models that are able to predict the water clog presence and the gas pore pressure increase.The instrumentation and measurements in PTM tests are very sophisticated. Today, PTM is considered to be the most advanced material testing method, helping the research community to observe the pore pressure development in fire conditions and to understand the physical mechanism behind it. This technique was also used to measure the effectiveness of PP fibre addition in pore pressure build-up in heated concrete and associate it with the spalling propensity of certain concretes. In The construction element in case of fire must present a load-bearing capacity for a certain time to allow for the evacuation of the building. Moreover, the fire brigade must conduct the intervention in safe conditions. In some situations, the spalling behaviour of concrete may jeopardise the safety of concrete works. For beams, slabs, and all tensile members, crack occurrence may lead to a release of gas pore pressure, which decreases the spalling risk. The surfaces that are directly exposed to fire and work under compression require verification with regard to the spalling of concrete cover. In Reference , it is s2, is used. Additionally, the nominal cover of the main reinforcement should be 40 mm. In the next method, a concrete mix should be used, to which it has been demonstrated experimentally that no spalling occurs under fire exposure. The following technique consists of the application of protective layers, to which it is demonstrated that no spalling of concrete occurs under fire exposure.For all structural elements made of HPC, and for concretes containing silica fume in an amount higher than 6% at the design stage, the risk of spalling should be taken into account. One of the following four methods should be applied. In the first prevention method, a reinforcement mesh, with a nominal cover of 15 mm consisting of wires of a diameter of 2 mm and a pitch size lower than 50 \u00d7 50 mm3 of concrete. Nevertheless, for a high PP fibre content, a mixing problem is observed, and homogeneity in the concrete mix is difficult to obtain. It was reported that an increase in the total volume of the cement paste content by about 15% enables the fibres to be covered with cement paste. The effectiveness of PP fibre addition, as a technological solution that prevents spalling, was confirmed by numerous experimental studies [3 of fibres is also recommended by the EUROCODE 2 [Other spalling prevention techniques have been tested on existing structures, such as the application of heat protection panels, made of wool or gypsum, that slow down the heating process, which is a commonly used solution . This ty studies ,28,29. MROCODE 2  standardPP fibres melt at a temperature of around 170 \u00b0C, i.e., lower than the temperature at which the maximum water vapour pressure in concrete is reached (190\u2013260 \u00b0C). In the market, low-temperature melting PB Eurofiber HPR (Heat Prompt Reaction) fibres can be found, and, for this fibre variation, melting occurs more quickly because their melting temperature is lower.2 and H2O. In At 350 \u00b0C, polypropylene burns and turns into COIn our study, we compared two PP fibres types, known as PP fibrillated polypropylene fibres and PB Eurofiber, with a low melting temperature, which have shown advantageous melting temperatures (162 \u00b0C compared to 170 \u00b0C for fibrillated PP fibres).After melting, the polypropylene is partly absorbed by the cement matrix. We have represented it in an experiment, in which several fibres are heated on a concrete disk. The thermocouple shows the temperature at the location of the fibres, as shown in Similarly, when fibres melt and are absorbed by a cement matrix, a network of pores is created, which increases the concrete permeability. This facilitates the transport of water vapour. The channels that remain after fibres have melted add up to the natural porosity of the cement paste and the porosity of the interfacial transition zone (ITZ) between the cement paste and aggregates.Nevertheless, as reported by Kalifa et al. , severalTwo hypotheses concerning this additional cracking in fibrous concrete were put forward. The first is that the fibres, while being heated, dilate by 7\u201310% , which cc = 100MPa), with different amounts of PP fibres (0\u20133kg/m3), the amount of 1.1 kg/m3 in concrete heated on one side leads to a pore pressure development of about 2 MPa, which is close to the one recorded on NSC . Pressure and permeability measurements suggest that a dosage of around 1 kg/m3 should be sufficient for this type of concrete and within this range of temperatures. This value may not be sufficient for other types of concrete, with a smaller porosity, as in UHPC [c = 40 MPa and fc = 60 MPa) exposed to moderate heating, the addition of 2 kg/m3 of PP fibres caused a three-fold reduction in the pore pressure [In order to better understand the thermo-hydric process leading to the spalling phenomenon and explain the role of polypropylene fibres in spalling mitigation, Kalifa\u2019s PTM experiment was employed. In this small-scale test, the influence of PP fibres on temperature distribution, pore pressure build-up, and mass loss in a heated specimen may be tested. According to the experiments conducted on concrete fc = 0MPa, witpressure .3 results in a 70% reduction in the value of the maximum pressure, which was observed when comparing it with a non-fibrous reference concrete. The incorporation of the fibres significantly reduces the values of the maximum pore pressures and decreases the temperature of the pressure peaks (180\u2013200 \u00b0C). It has also been noted that the effect of fibres does not increase significantly when the fibre content is higher than 1.75 kg/m3.Experimental investigations have demonstrated the effectiveness of fibres as a means of reducing the water vapour pressure in concrete. PP fibres at an amount of about 0.2% of the volume makes it possible to effectively reduce the internal pressures. The addition of 1.75 kg/mThere is an alternative explanation for the observed positive effect of PP fibres. Their introduction to concrete decreases the strength after melting by introducing a supplementary porosity that affects the strength. The reduced tensile strength may allow the formation of thermal cracks in the cross-section by unloading the thermal compression stresses necessary for spalling on the surface layer. This theory, highlighted in Reference , explain3, three different amount of PP fibres  and three lengths  were used. The five series of concrete were tested, designated as \u201cHPC_fibredosage_fibre length.\u201d The increase of the heated concrete gas permeability was measured using the Cembureau method, described in detail in Reference [The permeability of concretes with fibres increases rapidly with temperature. In Reference , differeeference . The stu3 does not strongly affect the initial properties of concrete . The test consisted of measuring the mass gain of the specimen, which is in contact with water. During the test, the water level remained constant, and an increase in the sample mass was recorded. The measurement set-up is shown in 3 of short PP fibres are presented, and their initial difference in strength was 15% (30 MPa).According to some sources, PP fibres at amounts of 0.1% to 0.2% lead to a decrease in the initial strength of concrete by 10% to 15%. The strength decrease is due to the lower stiffness of the fibres in the cementitious matrix. Moreover, the material compaction of the higher fibres\u2019 content is more complicated. Therefore, the concrete porosity may be the cause of the fibrous concrete mechanical performance decrease. The influence of the heating temperature on the changes in the compressive strength of the reactive powder concretes, with and without PP fibres, was studied , and the3 of fibres, are presented [c = 39.5 MPa), which confirms that the PP fibres contribute to the permeability increase.In resented . The appThe results for the compressive strength, tested at 120 \u00b0C, presents decreases of about 20%. This was explained by the presence of a hydral gradient in the middle of the specimen where the core of the specimen is saturated with water, and the surface has dried out. The observed decrease in the mechanical performance in the water clog zone is considered to contribute to the spalling behaviour. When the melting temperature of fibres is reached, water evaporation is possible. Therefore, for fibrous HPC and ordinary concrete, a partial strength restoration was observed at 250 \u00b0C. The concrete performance reaches 35% of the initial compressive strength, when the temperature reaches 600 \u00b0C for all tested materials.2. The concrete specimens in medium-scale tests may be reinforced and loaded or restrained concrete, depending on the laboratory procedure. Medium-scale tests are performed as a screening test to verify the behaviour of specific concrete mixtures in fire conditions and select the one that does not tend to spall. To investigate the influence of polypropylene fibres on concrete spalling behaviour in fire, the different testing methods were developed, and the most interesting method was described. To date, no standardised document concerning the testing of the spalling behaviour of concrete has been proposed to unify the material testing methods in order to allow for the screening of concrete mixes.The assessment of concrete behaviour at a high temperature is carried out using three types of testing methods at different levels of analysis: small-scale, medium-scale, and full-scale analysis. Small-scale tests serve to investigate the material behaviour of concrete exposed to an elevated temperature, i.e., the thermal strains, gas pore pressure, temperature gradient, or mechanical properties. Full-scale fire tests are carried out on full-sized concrete elements in a real situation, in which the boundary conditions, external load, and conditioning correspond to the design. A full-scale test, as the most representative test, shows the qualitative and quantitative behaviour of structural concrete elements subjected to fire. The medium-scale test is carried out on concrete slabs exposed to fire, with a surface area of ca. 1mc = 40MPa) with three amounts of polypropylene (PP) fibres\u20140.0, 0.45 and 0.90 kg/m3\u2014was investigated for two types of concrete of different water to binder (w/b) ratios: 30% and 40%. The specimens were heated in a laboratory furnace with the heating curve KS F 2257 [3, 0.05% in volume) significantly reduces the spalling behaviour in tested concrete, as shown in The assessment of the susceptibility of concrete to spalling can also be conducted using a simple small-scale test that relies on preparing laboratory samples , with and without PP fibres, and exposes them to an elevated temperature in a laboratory electric furnace. Such a test is commonly used to screen the concrete mix in order to check only if it spalls or if it is not under an elevated temperature. In those tests, samples are prepared with different types of fibres and dosages to optimise the minimum effective amount that reduces spalling and, at the same time, does not significantly affect fresh concrete properties and enable appropriate mixing and homogenization. A small, simple test using cylinders was carried out by Han . Normal-3. This research aimed to evaluate the aggregate size. A small, simple test on cylinders exposed to the ISO-834 [3 of PP fibres for each tested composition, as shown in Similarly, small samples were also tested by Li  using Ul ISO-834  heating These tests are not considered to be representative of a fire situation involving construction projects when the material is under load. In order to develop a test that is more representative of a fire situation and to investigate the behaviour of the material from loaded concrete samples, heating the setups for specimens under compressive loads are used. Such a test can be helpful for assessing the changes in the mechanical properties of concrete caused by an elevated temperature, as well as to study the PP fibre dosage effect on the change of properties. c = 98 MPa) under an elevated temperature and uniaxial compressive load action, with 2 kg/m3 and without the addition of PP fibres. Cylindrical specimens of \u00d8100 mm and H300 mm were heated at 1\u00b0C/min and loaded with a constant ratio. The setup enabled the measurement of the temperature changes by thermocouples, axial deformations by an extensometer, and internal damages by AE records, as shown in In several tests, an acoustic emission (AE) setup is additionally assembled near the specimen. Fire tests with an AE setup provide essential information on micro-cracking development that may differ for fibrous and non-fibrous concretes. Since micro-crack development is considered to enhance the moisture release and lower the pore pressure, information on it is essential for tests on the fire spalling of concrete. Huismann  tested cA simple weight loss technique was used to establish spalling events and sample mass loss during heating, as shown in the authors\u2019 research . The speIn 3 of fibre, spalling risk occurs when the heating rate is 4 \u00b0C/min, and 2 kg/m3 of fibres prevent spalling, even when 8 \u00b0C/min is applied , which were exposed to fire. In each case, the area exposed to fire was equal to 0.6 m \u00d7 0.6 m. For each tested case, the area exposed to fire was surrounded by an unexposed belt of different widths , which constituted a cold rim, as shown in The confinement to thermal strains in concrete can also be introduced by the presence of the unheated part of the concrete slab, which does not expand while heating. Unlike the steel ring, the unheated concrete part counteracts the expansion of the volume of the tested element that is exposed to inert fire. The authors have carried out the experimental tests on three different sizes of HSC slabs . By choosing the load level, both the restraint and additional external load may be introduced into the testing element. Commonly, the experimental setup consists of a gas furnace, with the opening on the top, on which the slab specimen is placed. Most often, squared slab-like elements are used for this kind of test. An external load is applied to the slab using hydraulic jacks, which are located on the sides of the slab, as shown in The effectiveness of the PP fibres in fire spalling mitigation of concrete elements may be evaluated because the external biaxial load prevents cracking. Thus, the load controls crack opening and moisture release.c \u00b1 60MPa) slabs, which were 0.8 \u00d7 0.8 \u00d7 0.1 m3 in size, were subjected to constant in-plane lateral compression of 10 MPa during the test. In one tested composition, the addition of 2 kg/m3 of PP fibres was used. Slabs were exposed to an ISO-834 fire scenario from the bottom side in an area of 0.6 \u00d7 0.6 m2. The test results are very similar to those obtained with the above-mentioned ring test. PP fibres do not prevent spalling but strongly reduce its occurrence and help protect the reinforcing steel. Both tests confirm that PP fibres can reduce spalling only to some extent and tests on spalling behaviour and the effective dosage of PP fibres, before designing concrete structures, are strongly recommended.The test, considering the effectiveness of PP fibres using loaded slabs, was presented by Lo Monte et al. in 2015 . The HSCThis paper presents an overview of current knowledge about the concrete spalling in fire and the role of polypropylene fibres in its effective mitigation. The main mechanisms leading to spalling occurrence include the thermal-mechanical and hygro-thermal mechanisms. The risk of concrete spalling arises together with an increase in concrete strength and density and is practically absent in low-grade concretes characterised by high degrees of open porosity. New-generation high-performance and ultra-high-performance concretes are particularly susceptible to the risk of fire spalling. The use of polypropylene fibres is recommended in order to limit spalling occurrence.Despite the numerous experiments conducted, the complex numerical hygro-thermal models developed. It is still not possible to effectively determine the risk of fire spalling for concrete with a given composition and properties. Spalling is a complex process that is affected by the composition of concrete  and its properties  and the spalling risk must be evaluated for a given application.The effectiveness of polypropylene fibres depends on their melting temperature, geometry length, and cross-section. We have shown that melting processes of fibres cause the creation of micro-cracks and contributes to the lower water vapour pressure. The melting temperature of PP fibres (170 \u00b0C) is very close to the peak measured by a number of experimental peaks of pore pressure created in a concrete section exposed to fire. Therefore, the addition of PP fibres to concrete provide successful results in reducing spalling risk due to fire.The quantity of fibres enabling the percolation of open pores in the matrix, which limits the gas pore pressure picks and prevents spalling, must be determined, and the experimental techniques are commonly used for this purpose. In addition, the manner in which concrete is loaded and the stress conditions during fire  plays an important role that needs to be considered when spalling risk is evaluated. Several examples of different approaches for assessing spalling risk in concrete due to fire were presented. It is undeniable that determining mechanical and physical properties of concrete under elevated temperature are vital in the design process. However, in the case of structures being at high risk of fire occurrence, it is recommended to provide the assessment of concrete susceptibility to spall due to fire exposure. The most appropriate approach for the fire test is to ensure the conditions that reflect the intended use of a targeted concrete element.The complex nature of the problem discussed, the absence of standard procedures for testing the susceptibility of concrete to fire spalling, and the stochastic nature of the process will undoubtedly result in further work on spalling and methods of spalling risk mitigation."}
+{"text": "Delirium tremens is a severe form of alcohol withdrawal syndrome. Literature documenting acute coronary events in the setting of alcohol withdrawal remains scarce. An accepted hypothesis for the underlying process is focused on the hyperadrenergic state that leads to coronary vasospasm and increased myocardial oxygen demand.A 47-year-old Caucasian man with a past medical history of tobacco and alcohol abuse, hypertension, and anxiety presented to the emergency department for crampy epigastric abdominal pain with intractable nausea and vomiting for the past 2 days. His reported last alcoholic intake was about 10 days prior; however, outpatient records indicated otherwise. He was admitted for electrolyte replacement and fluid resuscitation secondary to gastrointestinal losses from presumed early alcohol withdrawal syndrome. The following night, he developed acute substernal chest pain with elevated cardiac enzymes. Electrocardiography showed an acute inferoposterior infarct with reciprocal changes in leads V1\u2013V4. The patient was taken for emergent catheterization, and a drug-eluting stent was placed in the middle of the left anterior descending artery. Postcatheterization electrocardiography showed sustained inferolateral ST elevations consistent with acute injury pattern. The patient had not required any benzodiazepines until this point. On the morning of catheterization, the patient\u2019s Clinical Institute Withdrawal Assessment for Alcohol\u2013Revised score was 19 with a high of 25, and he was actively hallucinating. He was treated for delirium tremens and an acute coronary event along with an incidental pneumonia. He did not require any benzodiazepines during the last 4 days of admission, and he made a full recovery.The prevalence of alcohol dependence in hospitalized patients is substantial. Although our patient was being treated with the standard protocols for alcohol withdrawal, he rapidly developed delirium tremens, which led to an acute inferior ST-elevation myocardial infarction in the setting of nonoccluded coronary vessels. This case report adds to the sparse literature documenting acute coronary events in the setting of alcohol withdrawal and suggests that our patient\u2019s ST-elevation myocardial infarction is not fully explained by the current coronary vasospasm hypothesis, but rather was in part the result of direct catecholamine-associated myocardial injury. Further research should be conducted on prophylactic agents such as \u03b2-blockers and calcium channel blockers. Delirium tremens (DT) is a severe form of alcohol withdrawal syndrome that typically presents within 48\u201396\u2009hours and is defined by delirium, hallucinations, seizures, autonomic hyperactivity, and psychomotor disturbances . There hThe current hypothesis for the underlying process is the hyperadrenergic state that occurs during alcohol withdrawal leads to coronary vasospasm and increased myocardial oxygen demand , 3, 5, 6A 47-year-old Caucasian man with a past medical history of tobacco use, alcohol abuse, hypertension, and anxiety presented to the emergency department for crampy epigastric abdominal pain with intractable nausea and vomiting for the past 2 days. The patient also admitted to a chronic productive cough. The patient was brought in via emergency medical services after being orthostatic and tachycardic at the clinic earlier that day. The patient reported that his last alcoholic intake was about 10 days ago. However, outpatient records stated that the patient\u2019s last drink could have been anywhere from 1 week to 3 days prior. The patient was drinking about 1 pint of vodka per day and stated that he had undergone inpatient alcohol detoxification five or six times in the past.9/L and mild chronic transaminitis. He was admitted to the hospital for electrolyte replacement and fluid resuscitation secondary to gastrointestinal losses from presumed early alcohol withdrawal syndrome. Urine Streptococcus pneumoniae and Legionella antigen tests were ordered at that time. The patient was started on 1\u2009L of sodium chloride 0.9%, oral potassium chloride 20\u2009mEq twice daily, daily banana bags , home metoprolol 25\u2009mg twice per day, Clinical Institute Withdrawal Assessment for Alcohol\u2013Revised (CIWA-Ar) checks, seizure precautions, and ampicillin-sulbactam for suspected pneumonia.His vital signs upon admission showed a blood pressure of 158/111\u2009mmHg, pulse rate of 115 beats/minute, temperature of 37.2\u2009\u00b0C (99\u2009\u00b0F), respiration of 20 breaths/minute, and a peripheral capillary oxygen saturation of 99%. His physical examination showed mild epigastric tenderness to palpation but was otherwise unremarkable. Workup included a chest x-ray, which showed few air bronchograms projecting into the posterior lung base, procalcitonin of 0.61\u2009ng/ml, and lactate of 1.8\u2009mmol/L. The patient\u2019s sodium was 128\u2009mmol/L, potassium 2.34\u2009mmol/L, chloride 74.3\u2009mmol/L, magnesium 1.2\u2009mg/dl, glucose 202\u2009mg/dl, and serum alcohol <\u20090.010\u2009(g/dL). Laboratory tests showed a platelet count of 87\u2009\u00d7\u200910The following day, the patient\u2019s electrolyte imbalances had improved. Electrocardiography (ECG) showed sinus rhythm with left-axis deviation and poor R-wave progression Fig. . The patUp to this point, the patient\u2019s CIWA-Ar scores were 0. On the morning of catheterization, his CIWA-Ar score was recorded as 19 with a high of 25. The patient was actively hallucinating and required lorazepam, additional banana bags, and a 1:1 sitter. The patient did not develop seizures during this event. The patient was treated for an acute coronary event. A subsequent echocardiogram showed mild to moderate inferior hypokinesis at the base as well as anteroseptal and mild anterior hypokinesis with an ejection fraction of 50\u201355%. The patient developed atrial fibrillation with rapid ventricular response in the setting of his acute STEMI. The patient had a normal thyroid-stimulating hormone level and was rate-controlled with amiodarone and carvedilol. He was initially started on a statin, but this was discontinued after an unacceptable elevation of his liver function tests.S. pneumoniae antigen was positive the day following his acute STEMI, and his antibiotics were changed to intravenous ceftriaxone. He remained afebrile and never required supplemental oxygen. He made a full recovery and did not require any benzodiazepines during the last 4 days of admission. He stated that he was not interested in going back to rehabilitation and would prefer to quit on his own. The patient was discharged to home on day 7 with 10 days of amoxicillin-clavulanic acid, aspirin, clopidogrel, amiodarone, carvedilol, and Veteran Affairs follow-up.The patient\u2019s Anginal chest pain generally occurs only when there is greater than 70% stenosis of a major coronary vessel. Our patient denied any prior chest pain symptoms before his acute STEMI. Although he patient had many risk factors for developing an acute cardiac event, including incidental pneumococcal pneumonia, he had no known coronary artery disease. His elevated troponin level and his ECG suggested an inferoposterior infarct due to a right coronary artery (RCA) occlusion because approximately 85% of the population has right-sided dominance . HoweverTakotsubo syndrome [Although the patient\u2019s coronary vessels showed stenosis, there was no occlusion to explain his inferior STEMI. In this setting, there are several mechanisms that may explain what occurred. The patient could have had a thrombosis that occluded his coronary vessels and then spontaneously lysed prior to catheterization. Because there was no evidence of thrombosis or plaque rupture, this is unlikely. Although the current hypothesis of coronary spasm due to increased sympathetic hyperactivity remains a possible explanation, there was no documented vasospasm observed during catheterization. Additionally, the postcatheterization ECG showed sustained ST-segment elevations despite normal coronary flow Fig. . These Ssyndrome . The patBecause DT is an uncommon occurrence with current protocols, it is worth discussing how it might have developed in our patient. Our patient presented to the emergency department with intractable nausea and vomiting for 2 days. Although the patient\u2019s reported last alcoholic intake was questionable, his presenting gastrointestinal symptoms and laboratory test results were consistent with the mild symptoms of alcohol withdrawal syndrome. The patient\u2019s laboratory test results and clinical state were improving with initial resuscitation. He maintained CIWA-Ar scores of 0 and required no benzodiazepines within the first 24\u2009hours. It was difficult to identify the exact timeline of when the patient developed DT and the STEMI because they seemed to occur simultaneously. As the patient\u2019s cardiac issues were given priority, the first rising CIWA-Ar score was not recorded until after catheterization, when the patient required benzodiazepines. However, nursing staff reported that the patient became more irritable and was ripping off his bed covers, looking for his \u201ckey fob\u201d in reference to his call light, prior to developing chest pain, suggesting that his delirium preceded his cardiac event.DT typically does not occur suddenly; rather, it progresses in a more sequential timeline . Our patThe prevalence of alcohol dependence in hospitalized patients has been shown to be about 16\u201326% and up to 42% in hospitalized veterans . Approxi"}
+{"text": "Odontogenic cysts are usually treated by enucleation (cystectomy). Limited cysts (less than 5\u2009cm) are usually managed by primary excision , whereas larger ones (exceeding 5\u2009cm) are often decompressed or marsupialized. Because it consists only of opening a much smaller surgical window, decompression is regarded as a more conservative method of treatment: this method associates the creation of an opening (window) into the cystic cavity with the suturing of a decompressing device (plastic tube or stent) at the periphery of the cyst. Apart from releasing intraluminal pressure in the pathological cavity, this procedure helps the lesion to progressively decrease in volume \u201cwith a gradual increase in bone apposition\u201d and preserves pulp vitality and periodontal integrity of the adjacent teeth. We are reporting a case of a mandibular radicular cyst that was treated by decompression, followed by enucleation, bone reconstruction, and restoration with two osseointegrated dental implants. The cystic cavity progressively decreased in volume and increased in bone density. Marsupialization of odontogenic cystic lesions was described by Partsch in 1892; it is a technique where a large window is made in cystic wall and then sutured to the oral mucosa , it's baBone defects can occur inevitably after mandibular cyst enucleation. A bone graft can be planned to promote bone regeneration of this bony defect , 6. The The aim of the present study is to present a clinical case report of a radicular cyst in a female patient that was treated by decompression, followed by cyst's enucleation, bone reconstruction of the cyst cavity, and dental implant placement and restoration.A 45-year-old female patient consulted our office with a chief complaint of left facial swelling and numbness of her chin, left lower lip, and a part of her left buccal mucosa and mandibular posterior vestibule. The patient had a history of endodontic treatment of her mandibular second left premolar (tooth 35) that was not accomplished by her restorative dentist; she also revealed that the swelling increased and reached her mid lower face, extending to the left anterior triangle of her neck, as well. She was treated by her restorative dentist with intramuscular injections of Rocephin\u00ae (ceftriaxone) during 4 weeks without remission.Patient originally consulted for left facial swelling and paresthesia of left lower lip and chin. The left posterior mandibular vestibule, and particularly apical regions of 34 to 37, was spontaneously painful.Intraoral examination displayed two swellings (in the left mandibular vestibule in relation to teeth 35 and 36) that were tender to palpation. On clinical examination, teeth 35 and 36 showed extreme mobility and were painful on horizontal and vertical percussion. Tooth 35 was open to access pulpal cavity, and thermal pulp test was negative on teeth 34 and 36 and positive on tooth 37.An orthopantomogram (OPG) was taken in order to determine a possible bony pathology underneath the swollen left mandibular vestibule: OPG displayed a well-limited radiolucent image, extending from tooth 34 to tooth 37 and occupying most of the mandibular basal bone, in this sector. Periapical radiolucent lesion measured around 3-4\u2009cm in length with around 2\u2009cm width. It appeared unilocular, with well-defined, nonsclerotic borders, extending from mesial aspect of 34 to mesial aspect of mesial root of tooth 37, and almost reaching lower border of the mandible. Aspiration of the lesion was performed under local analgesia and it released pus and blood and a presumptive diagnosis of infected radicular cyst was made Figures .Axial and coronal cuts of a cone beam computed tomography (CBCT) showed a radiolucency, ball shaped, measuring approximately 30.7\u2009mm \u00d7 19.7\u00a0mm with discontinuity of the buccal wall, and the mandibular canal is repressed toward the basic bone of the mandible Figures .Treatment plan was decided and discussed with the patient, and the agreement was to undertake root canal treatment of tooth 34 and extract teeth 35 and 36 and to perform decompression of the radiolucent lesion through their alveolar cavities, after extractions of these teeth.Block analgesia of the left inferior alveolar nerve was implemented with block 2% articaine with 1\u2009:\u2009100,000 adrenaline ; teeth 35 and 36 were extracted using elevators and forceps.An incision through alveolar processes of 35 and 36 was made with no. 15 blade and the lesion's lining was fenestrated with a scalpel's blade: two transparent plastic tubes, sectioned from the tubing of a standard saline bag, were inserted in each alveolar cavity, and two interrupted sutures, from each side of the tubes, were placed to stabilize them Figures .The patient was instructed to self-irrigate the lesion with a plastic disposable syringe with needle, with two antiseptic solutions, normal saline solution (0.9% sodium chloride in water) during the day  and 0.12% chlorhexidine gluconate in the morning and in the evening before bedtime.The patient was recalled every week for irrigation of the lesion and check-up. What is special in this irrigation modality is that the antiseptic solution  was injected through one tube and drained from the other one, instantaneously. At biweekly intervals, the length of the tube was monitored and cut in order to keep it far from occlusion .The lesion's enucleation was planned to be implemented 6 months after decompression that would reduce its size. Indeed, 6 months after the first lesion's irrigation, a panoramic radiograph was taken in order to evaluate the bone apposition: bone formation took place within the lesion as observed on CBCT performed after 7 months, with an almost complete formation of buccal cortical and bone apposition buccally (\u00b14\u2009mm) and lingually (\u00b12.4\u2009mm) as shown on the para-axial cut and lesion volume reduction as shown on the sagittal reconstruction (\u00b125.1\u2009mm \u00d7 15.2\u2009mm), all along the lesion Figures .At first week of the 7th postoperative month, the decompression tubes were removed. With a blade #15, a crestal incision followed by 2 relaxing incisions was performed before raising a full-thickness flap. After separating the lesion's membrane from the flap, enucleation was accomplished with a Lucas curette and the entire pathological specimen was collected and immersed in a fixative solution (20% formol) for histopathological examination.During operation, it was noticed that the buccal bone wall took the same shape of drainage tubes after reformation. After the lesion's enucleation, the bony cavity was filled with Puros\u00ae cortico-cancellous particulate allograft  due to its osteoconductive properties. The flap was relocated with 5/0 nylon interrupted sutures, and tube opening locations were covered with collagen (CollaTape\u00ae) over bone graft Figures .The patient consulted us again one week after the cyst enucleation with less than 20% of left lower lip/chin numbness remaining, and two weeks later, she almost recovered her normal sensation, with a mild left lower lip \u201cheaviness\u201d remaining Figures .Pathological report concluded that the lesion was a radicular cyst .Six months following bone grafting, CBCT shows a complete bone regeneration of the whole area  and two Radicular cyst is the most frequent odontogenic cyst observed in tooth-bearing areas: it is usually associated with carious, nonvital, discolored, or fractured tooth. Dental caries are known to provocate dental pulp inflammation that leads, ultimately, to pulp necrosis; infection will then spread to periradicular space, causing periodontitis that usually precedes either periradicular acute abscess, chronic granuloma, or radicular cyst. This cyst usually follows persistent chronic infection, and it is believed to form by proliferation of the epithelial cell rests of Malassez in inflamed periradicular tissues .Several treatment options are nowadays available for a radicular cyst such as nonsurgical root canal therapy (endodontic treatment), extraction of the offending tooth if unrestorable, decompression, marsupialization, and enucleation with primary closure, when the lesion is large . The treDecompression procedure is \u201can alternative plan of treatment and a more conservative approach; this procedure allows for continuous drainage, which removes those conditions that favor cyst expansion\u201d . In the Decompression devices (tubes) aim to maintain drainage of jaw pathological lesions and facilitate their repeated irrigations : in our Decompression tubes were immersed in 0.12% chlorhexidine gluconate for 15 minutes and then sterilized before placement in the pathological cavity. They were inserted using a slight rotary motion and gently pushed downwards into the depth of the lesion until resistance was met at the lesion's lower level. Initial drainage occurred immediately after their insertion.It is widely known that decompression and marsupialization are conservative treatment modalities that involve an opening to reduce intracystic pressure and induce bony formation , 3, 12: In our case, decompression led to reduce the volume of the cyst from 30.7\u2009mm \u00d7 19.7 mm to 25.1\u2009mm \u00d7 15.2 mm approximately. The CBCT shows a complete bone formation of the cortical buccal plate and bone apposition from the buccal bone and lingual bone in a central direction measured as \u00b14\u2009mm and \u00b12.4 respectively.The effects of decompression based on age are controversial: it was reported that younger patients had higher reduction rates , but it Another advantage of these two methods, particularly decompression, is the positive effect they have on the histological nature of the cyst's epithelial lining. In a study on 14 decompressed odontogenic keratocysts (OKCs) with 65% of mean shrinkage of the radiolucency, August and coworkers reported a dedifferentiation and loss of cytokeratin-10 production in 64% of decompression and irrigation patients after a 9-month average treatment time: changes in cystic epithelium appeared to be a gradual process and their results suggested that a treatment time of at least 9 months may be required to induce epithelial dedifferentiation .Schlieve and coworkers  studied The most important requisites for bone deposition in a bone cavity includes the presence of blood clot, a source of osteoblasts, a contact with living tissue, and prevention of soft tissue invasion . There hIn our case, after the cyst's enucleation, we grafted the cavity with allograft, an osteoconductive material , knowingRadicular cyst is commonly found in the jaws. This case report illustrates the successful management of a mandibular radicular cyst by decompression, enucleation, grafting, and site rehabilitation."}
+{"text": "Depression of the Nrf2/CBR1 pathway in both mothers and their offspring is one of the causes of oxidative stress leading to MetS. This study suggests that 25-hydroxyvitamin D treatment may relieve the offspring's MetS.Metabolic syndrome is a disorder of energy use and storage, which is characterized by central obesity, dyslipidemia, and raised blood pressure and blood sugar levels. Maternal 25-hydroxyvitamin D de\ufb01ciency is known to cause metabolic changes, chronic disease, and increased adiposity in adulthood. However, the underlying mechanism of induced metabolic syndrome (MetS) in the offspring in vitamin D deficient pregnant mothers remains unclear. We identified that maternal 25-hydroxyvitamin D deficiency enhances oxidative stress, which leads to the development of MetS in the mother and her offspring. Further, immunohistochemical, Western blotting, and qRT-PCR analyses revealed that maternal 25-hydroxyvitamin D deficiency inhibited the activation of the Nrf2/carbonyl reductase 1 (CBR1) pathway in maternal placenta, liver, and pancreas, as well as the offspring's liver and pancreas. Further analyses uncovered that application of 25-hydroxyvitamin D activated the Nrf2/CBR1 pathway, relieving the oxidative stress in BRL cells, suggesting that 25-hydroxyvitamin D regulates oxidative stress in offspring and induces the activation of the Nrf2/CBR1 pathway. Taken together, our study finds that maternal 25-hydroxyvitamin D deficiency is likely to result in offspring's MetS probably  It has long been recognized that maternal nutrition along with environmental or physical conditions, has large impacts on the intrauterine environment, and ultimately, the fetal epigenome, including immune and metabolic functions in offspring . David BMetS is an assortment of cardiometabolic diseases and diabetic risk factors that has emerged as a global epidemic characterized by abnormal lipid metabolism, oxidative damage, IR and/or glucose tolerance . MetS isvia manipulation of target genes expression levels. A recent study from via the upstream ARE region (via antioxidation.The nuclear factor erythroid 2 related factor 2 (Nrf2) is an important factor in cellular defense function that manages cell response to oxidative stress . Recent E region , we propvia suppressing the Nrf2/CBR1 pathway. The study aimed to investigate the effects and mechanism of maternal 25(OH)D restriction on metabolism in adult offspring.In this paper, we hypothesize that maternal 25(OH)D deficiency or restriction during pregnancy has the potential to impact the metabolism of the offspring All tissue samples were collected from animals described as follows. All protocols were approved by the Ethics Committee of the Laboratory Animal Centre, Wenzhou Medical University (no. wydw 2018-0031).Sprague-Dawley female and male rats  were purchased from SLRC laboratory animal Co., Ltd . Animals were housed in a temperature-controlled room (constant temperature of 21 \u00b1 2\u00b0C and a humidity of 60 \u00b1 10%) with 12 h light/12 h dark rhythm cycle, and had free access to food and water. Female rats were randomly divided into two groups: a 25(OH)D deficiency group (VDD) which was given a 25(OH)D free diet (AIN-93G without 25(OH)D; AIN-93G, Keaoxieli Fodder, Beijing, China) and housed under incandescent light (free of ultraviolet radiation), and a control group (Con) which received a 25(OH)D-replete diet D concentration = 1,000 IU/Kg) and normal light (with ultraviolet radiation) for 6 weeks. To encourage mating, the female rats were placed overnight with the male rats. Embryonic day 0 (E0) was defined as the day a sperm-positive vaginal smear was obtained. Pregnant rats (VDD-F0) was remain given a 25(OH)D free diet, and pregnant rats (Con-F0) received a 25(OH)D-replete diet throughout their pregnancy.Pregnant rats were all individually housed. We defined age week 0 as the first day with the appearance of pups. Following birth, all male pups from both Con-F0 and early-VDD groups were immediately removed and suckled by dams in the control group and consequently placed under control light conditions. The male offspring were standardized to eight per litter. Once male offspring were weaned, all males were housed in individual cages and fed a 25(OH)D-replete diet, until 16 weeks of age.To investigate any metabolic programming changes and expression levels of proteins of interest in the offspring, we chose four time points: 0 week (birth), 4 weeks (weaning), 8 weeks (near adulthood), and 16 weeks (adulthood). At each of these time points, ten offspring were selected from different litters (chosen randomly from different groups) and were weighed, fasted overnight, and anaesthetized. Blood was collected and then the pups were sacrificed by decapitation. The tissues were removed, weighed, snap-frozen in liquid nitrogen, and stored at \u221280\u00b0C. The placenta, liver, and pancreas sections that were designated for histology were rapidly formalin-fixed and paraffin embedded for immunohistochemistry analysis.To detect ROS levels, tissue samples were pretreated before performing flow cytometry analysis. For pretreatment, adequate amounts of fresh tissues were cut up and 0.125% trypsin solution was added and incubated for 30 min at 37\u00b0C for tissue digestion. Tissue digestion was terminated with serum-containing medium. The cell suspension was transferred through a 200 mesh stainless steel screen, centrifuged at 402 (\u00d7g) for 5 min at 4\u00b0C. After centrifugation, ROS level was determined using flow cytometry.To determine the protein concentrations in tissue samples, rat liver, placental, and pancreatic tissues were homogenized on ice using the radioimmunoprecipitation assay , followed by centrifugation at 16,200 (\u00d7g) for 15 min at 4\u00b0C. The supernatants were tested for the levels of superoxide dismutase (SOD) and protein levels were determined by Western blot analysis.Tissues obtained for immunohistochemical (IHC) analysis were immediately fixed in 10% (v/v) neutral buffered formalin. Each specimen was then embedded in a paraffin block to prepare for IHC assay.2 incubator at 37\u00b0C. Only cells in the logarithmic phase from the third to eighth passage were used for experiments.The rat liver cell line (BRL cell) was purchased from the Cell Bank of Shanghai. Cells were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum (FBS)  in a humidified 5% COCBR1-specific small interfering RNA (siRNA)  were used to knock down CBR1. Cells were seeded in 6-well plates (2 \u00d7 105 cells/well) and culture overnight in DMEM medium supplemented with 10% FBS without antibiotics. Cells were then transfected with either CBR1 siRNA or a nonsilencing control siRNA using an siRNA reagent system (GenePharma) according to the manufacturer's protocol. Twenty-four hours after transfection, Western blot analysis was performed to detect the knock down efficiency of CBR1.CBR1-specific siRNAs were effective at knocking down CBR1 with the siC1-1 to be the most robust one. Following transfection, the cells were exposed to various treatments and analysis as described below.All the 2D3 , we treated BRL cells with 1,25-(OH)2D3 at a series of concentrations  for 24 h, and then the Nrf2 protein levels were determined by Western blot analysis.To identify the most effective concentration of 1,25-(OH)To determine the most effective concentration of Brusatol , BRL cells were treated with different concentrations of Brusatol  for 24 h, and then the Nrf2 protein levels were determined by Western blot analysis.2D3 , 1,25-(OH)2D3  + Brusatol (160 nM), or 1,25-(OH)2D3  + siC1-1. Nrf2 and CBR1 expression levels were measured by Western blot and real-time PCR.After initial experiments, cells were incubated under different conditions: 1,25-(OH)BRL cells treated with or without VitD3 for 48 h were used for the cross-linking with formaldehyde. For the immunoprecipitation, normal rabbit IgG and anti-Nrf2 antibody  were added respectively and incubated at 4\u2103 for 4 h. After DNA extraction, the CBR1 promoter region was amplified by PCR using the following primers:Forward: 5\u2032-GTTCTTCACTGCCACGTG-3\u2032Reverse: 5\u2032-TGAACCATCCCTACCCCTC-3\u2032.The serum levels of triglycerides (TG), high-density lipoprotein (HDL-C), fasting blood glucose (FBG) and insulin (INS) were assayed using the appropriate assay kits. The FBG biochemical kit was purchased from Applygen . The INS, TG, and HDL-C ELISA kits were purchased from Bioswamp .The serum samples were harvested and used for examining related indexes according to the manufacturer's protocols. Serum chemistry levels were measured using an automated analyzer .Homeostasis model assessment (HOMA) models were used to estimate IR (HOMA-IR), insulin sensitivity (HOMA-S), and \u03b2-cell function (HOMA-\u03b2), which were calculated by the following equations: HOMA-IR = [fasting insulin (mU/L) \u00d7 fasting glucose (mmol/L)]/22.5, HOMA-S = 22.5/[fasting insulin (mU/L) \u00d7 fasting glucose (mmol/L)], and HOMA-\u03b2= [20 \u00d7 fasting insulin (mU/L)]/[fasting glucose (mmol/L) \u2212 3.5].C1-1, all cells were digested with 0.25% trypsin, and 500 \u03bcL of 1 \u03bcM CM-H2DCFDA  were added to each tube and incubated at room temperature in the dark for 30 min. Next, cells were washed twice with phosphate-buffered solution (PBS) and gently resuspended in DMEM medium containing 10% FBS, and then analyzed using flow cytometry . For the tissues, single cell suspension prepared from placenta, liver, and pancreas tissues of rats.To confirm the levels of ROS in BRL cells after treatments with Brusatol, VD3, or siThe harvested protein supernatants were used to evaluate the levels of SOD in tissues by following the SOD detection kit protocol provided by the manufacturer .Maternal placentas were immersed in 4% paraformaldehyde and incubated for 4 h, and then transferred to70% ethanol. Individual lobes of placenta biopsy material were placed in processing cassettes and dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks. Before staining, 5-\u00b5m thick placenta tissue sections were dewaxed in xylene, and rehydrated through decreasing concentrations of ethanol, and washed in PBS. The sections were then stained with H&E.Nrf2 , CBR1 , IL-1\u03b2 , IL-6 , and TNF\u03b1  were used for the IHC detection of protein expression in the trimethylamine (TMA). Nrf2 and CBR1 antibodies were both used at a 1:200 dilution. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. The TMA samples were incubated with biotinylated antibodies and horseradish peroxidase. Staining was developed with diaminobenzidine substrate and the sections were counterstained with hematoxylin. Negative controls were obtained by using PBS instead of Nrf2 or CBR1 antibody. The expressions of Nrf2 or CBR1 were quantified as previously described .\u00ae chemiluminescence reagents (Amersham Biosciences)according to the manufacturer's instructions, followed by exposure to X-ray \ufb01lms (Sigma Z370398). Films were analyzed with Quantity One Software (BioRad Laboratories), and the resulting absorbance values were expressed as the percentage variation of the control group values. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.The protein concentrations of the samples were measured using a BCA protein assay kit . The samples containing equal amounts of proteins (30 \u03bcg) were denatured in sample loading buffer and separated by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis, followed by a transfer to a nitrocellulose membrane . Then membranes were blocked with 5% (w/v) non-fat dried milk in a Tris-buffered saline with Tween 20 (TBST), and incubated with primary antibodies  and anti-CBR1 ) overnight at 4\u00b0C. Then membranes were washed three times and incubated with horseradish peroxidase-conjugated anti-goat (for Nrf2) or anti-rabbit (for CBR1) second antibodies  for 2 h. Immunoreactive bands were visualized using ECLNrf2 and CBR1 amplification signals were normalized to GAPDH expression. Gene-specific primer sequences used for qRT-PCR are listed as follow:Total RNA was extracted from various rat tissues using RNA extraction kit  according to the manufacturer's instructions, and used for reverse transcription into cDNA by applying RevertAid First Strand cDNA synthesis Kit . The cDNA was then subjected to RT-qPCR analysis using SsoAdvance Universal SYBR Green Supermix , and gene expression was quantified as previously described . The NrfCBR1 Forward: 5\u2032-TTGACGAGACCTACCTGATGTT-3\u2032;Rat CBR1 Reverse: 5\u2032-TGTGGATGATGATGCTCTTCTG-3\u2032.Rat Nrf2 Forward: 5\u2032-GAGACGGCCATGACTGAT-3\u2032;Rat Nrf2 Reverse: 5\u2032-TGAGGGGATCGATGAGTA-3\u2032.Rat GAPDH Forward: 5\u2032-GGTGGACCTCATGGCCTACA-3\u2032;Rat Rat GAPDH Reverse: 5\u2032- CTCTCTTGCTCTCAGTATCCTTGCT-3\u2032.t-test for two experimental groups and two-way ANOVA for multiple groups were employed to assess statistical differences. A value of P < 0.05 denotes statistical signi\ufb01cance, P < 0.01 denotes a higher level of signi\ufb01cance and P < 0.001 denotes a very high level of statistical signi\ufb01cance. Statistical analyses were done using GraphPad Prism 6 .Results are expressed as means \u00b1 SD. The unpaired 2-tailed Student's To determine the validity of the model, the maternal vitamin D level was measured on embryonic day 0 (E0) and embryonic day 21 (E21). As shown in in vivo, we chose to examine several metabolic indicators, including blood TG levels, HDL-C levels, fasting insulin (INS) levels, and the FBG levels of pups from 25(OH)D deprived pregnant rats. We found that the levels of TG, FDG, and INS were significantly increased in the 25(OH)D deprived pups D deficiency may be mediated through several different mechanisms, including increased oxidative stress and IR . To explved pups Table 1.P < 0.001) and amniotic epithelium had more branches and a larger amount D deficiency rat model induced reduction in Nrf2 and CBR1 protein expression in the maternal placenta, liver, and pancreas D deficiency rat offspring at week 0 and week 16 D induced upregulation of Nrf2 and CBR1 expression D on the Nrf2/CBR1 pathway, we treated BRL cells with 25(OH)D and examined the Nrf2 expression levels. BRL cells treated with 25(OH)D demonstrated a significant upregulation of Nrf2 expression levels, and this effect was found to be 25(OH)D dose dependent D and oxidative stress. Treatment of BRL cells with 25(OH)D caused a significant increase in SOD production. This increase was inhibited by co-treatment with Nrf2 inhibitor Brusatol, or siHere, we reveal evidence that maternal 25(OH)D deficiency beginning around the time of conception can cause MetS in the adult offspring, and that this may be induced by higher levels of oxidative stress in the offspring. We observed increased levels of TG, FBG, INS, and HOMA-IR, and decreased level of HDL-C in peripheral blood of the offspring from mothers that experienced 25(OH)D deficiency during pregnancy Figure 1CBR1.To explore the role of oxidative stress in promotion of MetS, we examined the Nrf2/CBR1 pathway, which is known to play a role in anti-oxidative stress processes. Takeshi Miura et al. identified that Nrf2, a novel transcriptional regulator, regulates the transcription of antioxidant enzymes, and CBR1, an antioxidant enzyme, is regulated by Nrf2 . PreviouIn light of Nrf2 and CBR1 expression reduction in 25(OH)D deficient maternal placenta Figure 4via vitamin D receptor to ameliorate leptin-induced oxidative stress D status around the time of conception has been found to be closely related to many health outcomes in mother and offspring. Numerous studies reported the associations between 25(OH)D status in pregnancy and a variety of obstetric complications, including gestational hypertension, pre-eclampsia, and gestational diabetes (GDM) . Three se stress . These rHowever, there were some limitations in our study that still need to be thoroughly investigated. First, because the details of metabolic disorder are extremely complex, the molecular mechanisms of metabolic disorder regulated by 25(OH)D should be examined outside of the role it plays in the Nrf2/CBR1 pathway. Also, considering Nrf2 is a transcription factor, further clarification is needed to determine if Nrf2 regulates the transcription of other antioxidant genes, in addition to CBR1. In addition, although our study unveiled the influence of maternal 25(OH)D deficiency on offspring, different degrees of influence on MetS may result from maternal 25(OH)D deficiency in different genders of offspring and a lack of glucose tolerance test is one limitation in our study. Hence, understanding how the gender of the offspring influences the response to maternal 25(OH)D deficiency still remains to be explored.via abnormal nutrition transformation across placenta. Furthermore, the depression of the Nrf2/CBR1 pathway in both the mother and her offspring is one of the cause of oxidative stress leading to MetS. This study suggests that 25(OH)D treatment may relieve the offspring's Mets.In conclusion, in our study, we find that maternal 25(OH)D deficiency is likely to result in offspring's MetS probably The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Ethics Committee of the Laboratory Animal Centre, Wenzhou Medical University.HY and JQZ contributed to the study design. XL, BZ, ZZZ, and HZ collected data. JYZ, XX, and CS analyzed data. HC, JY, and ZW interpreted the results. ML, JC, QZ, ZZ, and JQZ wrote and revised the final draft of the manuscript.This work was supported by the Natural Science Foundation of Zhejiang Province under Grant No. LY13H040010, LQ18H040002.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Today\u2019s healthcare organizations want to implement secure and quality healthcare software as cyber-security is a significant risk factor for healthcare data. Considering security requirements during trustworthy healthcare software development process is an essential part of the quality software development. There are several Security Requirements Engineering (SRE) methodologies, framework, process, standards available today. Unfortunately, there is still a necessity to improve these security requirements engineering approaches. Determining the most suitable security requirements engineering method for trustworthy healthcare software development is a challenging process. This study is aimed to present security experts\u2019 perspective on the relative importance of the criteria for selecting effective SRE method by utilizing the multi-criteria decision making methods.The study was planned and conducted to identify the most appropriate SRE approach for quality and trustworthy software development based on the security expert\u2019s knowledge and experience. The hierarchical model was evaluated by using fuzzy TOPSIS model. Effective SRE selection criteria were compared in pairs. 25 security experts were asked to response the pairwise criteria comparison form.The impact of the recognized selection criteria for effective security requirements engineering approaches has been evaluated quantitatively. For each of the 25 participants, comparison matrixes were formed based on the scores of their responses in the form. The consistency ratios (CR) were found to be smaller than 10% (CR\u2009=\u20099.1%\u2009<\u200910%). According to pairwise comparisons result; with a 0.842 closeness coefficient (Ci), STORE methodology is the most effective security requirements engineering approach for trustworthy healthcare software development.The findings of this research study demonstrate various factors in the decision-making process for the selection of a reliable method for security requirements engineering. This is a significant study that uses multi-criteria decision-making tools, specifically fuzzy TOPSIS, which used to evaluate different SRE methods for secure and trustworthy healthcare application development. Today\u2019s Information Technology (IT) has had a massive impact on a number of areas of society. We live in an era of IT in which technological resources such as software, hardware, and sensors are becoming an essential accessory in our daily lives , 2. As wThe security violation for a healthcare software product causes enormous fatalities. For this reason, it is necessary to develop such a security-critical software system in the best conceivable way. It does not inevitably mean absolute security, however a reasonable high-security level in relation to the given limitations. In recent years, literature has offered a number of security and privacy requirements engineering methods that assist the software system designers and developers to implement security and privacy concerns presented in the traditional development model. Several methods deliberate security or privacy requirements independently, however, some other approaches consider privacy as a subset of security . SoftwarIn the United States in 1996 the Health Insurance Portability and Accountability Act, known as HIPAA, was enacted. The law sets standards for data security as well as privacy to safeguard patient records. HIPAA compliance has now become an important consideration in the healthcare industry for software engineers in recent times, as several high-profile data attacks have exposed millions of medical information nationally . AccordiSoftware security is the fastest growing paradigm in the IT security field , 8. AccoSecurity requirements are one of the most important parts of all non-functional requirements. Negotiation with software security requirements during software development may result in disastrous failure of the software product affecting enormous damage of valuable assets. Special attention should be given to the security requirements of the software product during the software development as a software system does not exist physically.The introduction of electronic health records raises a variety of problems related to protection and privacy that need addressing. Cyber-security in the healthcare sector has been one of the major challenges. Recent initiatives to digitize various dimensions of healthcare, the transition to electronic health, would have a huge effect on healthcare sector growth. Healthcare records and information are distributed across networks, which mean they are sensitive and vulnerable to a range of security breaches. The possible threats for healthcare breaches may include medical staff, malware or phishing attacks, healthcare suppliers, electronic health equipment, unauthorized access, etc. . There aObviously, the biggest challenge is to avoid improper access or misuse of the medical records. The next challenge is whether any permitted access is traceable. This in effect includes procedures for safe identification of individuals including doctors, patients, labs, etc. Eventually, documents in a censored or anonymized form ought to be made accessible to third parties. The rising threat to healthcare and patient\u2019s confidential data enables application developers to become more rigorous in designing successful security requirements to make trustworthy healthcare web applications. For many healthcare application developers, security requirements are a major concern, and some of those have vital role in the proper organizational goals of the software development will try to incorporate security into the application development process as soon as possible.The identification of threats is significant and it facilitates the development of realistic and relevant security requirements. Implementations for healthcare services should be capable of storing and handle claims for refund. In addition, the database would be open to enable access to information about patient treatment in the event of immediate necessity. There\u2019s really common understanding between many practitioners and researchers that security effective and efficient security requirements elicitation is important. Therefore the creation of healthcare applications requires the integration of functional, non-functional requirements and also architectural engineering practices . One chaWe notice that different security requirements engineering techniques are available for the elicitation of security requirements in order to develop a quality and trustworthy healthcare software system. However, the selection of the most appropriate SRE approach for trustworthy healthcare software development is a challenging task. The objective of this paper is to identify the different criteria for the analysis of different security requirements engineering approaches. Further, we select the ISO 27005 standard  criteria\u201cSecurity requirements engineering is a process of generating prerequisite non-functional requirements that stipulate a compulsory amount of system quality to prevent the adversary\u2019s attack. It should be considered early during the software development process to deliver a trustworthy software product.\u201dSecurity requirements engineering is an area of software engineering, which comprises security, safety, risk, vulnerabilities, and mitigation mechanisms. Security requirements engineering has over the years proven to be a challenging task. This is especially the case because pinpointing what security requirements are having been difficult. Despite all these challenges, the demand for developing security requirements elicitation methods for the changing requirements of networked environments is great. Mellado et al.  describeSoftware security engineering procedure should include the use of repeatable and organized processes to guarantee that the set of requirements found is complete, reliable, easy to recognize and analyzable by the different participants involved in the software development process . SecuritMellado et al. proposed Security Requirements Engineering Process (SREP)  is a metI.Agree on DefinitionsII.Identify Vulnerable &/or Critical AssetsIII.Identify Security Objectives & DependenciesIV.Identify Threats & Develop ArtifactsV.Risk AssessmentVI.Elicit Security RequirementsVII. Categorize & Prioritize RequirementsVIII. Requirement InspectionIX.Repository ImprovementSREP approach includes the following steps:I.Agree on definitionsII.Identify security goalsIII.Develop Artifacts to support security requirements definitionIV.Perform risk assessmentV.Select elicitation techniquesVI.Elicit security requirementsVII. Categorize requirementsVIII. Prioritize requirementsIX.Requirements inspectionSQUARE   developeThe SQUARE method had been modified to handle privacy (P-SQUARE) as well as acquisition (A-SQUARE) explicitly.I.Identify System GoalsII.Identify and Prioritize StakeholdersIII.Agreed upon GoalsIV.Asset IdentificationV.Point of Attack (PoA)Point of Belief (PoB)Point of Conjecture (PoC)Point of Dependency (PoD)Security Attack AnalysisVI.Threat Identification and CategorizationVII. Risk Evaluation and PrioritizationVIII. Security Requirements ElicitationIX.Security Requirements ValidationX.Security Requirements Specification DocumentAnsari et al. developed Security Threat Oriented Requirements Engineering Methodology (STORE) Methodology which is a ten-step security threat centric security requirements engineering methodology . It provI.Inception: Recognize web app goals, stakeholders as well as assetsII.ElicitationIII.Elaboration: Produce structural analysis modelsIV.Negotiation and validation of requirementsThe Model Oriented Security Requirements Engineering approach  seeks toHaley et al. proposed Security Requirements Engineering Framework  which isI.Identify functional requirementsII.Identify security goalsIdentify assetsGenerate threat descriptionApply management principles III.Identify security requirements: Constraints on some or all of the security objectives. The safety requirements are descriptively represented.IV.Construct satisfaction arguments: represent that the application is capable of meeting security requirements.To make good decisions we must well prioritize the recognized ISO 27005 standard criteria for effective security requirement engineering selection, and so it is important to take the consideration of all 25 security experts into account when prioritizing. In our prioritization process we use the technique of pairwise comparison of different criteria through fuzzy TOPSIS. In order to selection of best SRE approach; we need to compare pairs of these criteria.The first step in this process is the establishment of a hierarchy model. The hierarchy model is composed of the seven criterion group Security goal, Security requirement, Stakeholder, Asset, Threat, Vulnerability, Risk. After the hierarchy has been established, the criteria must be evaluated in pairs so as to determine the relative importance between them and their relative weight to the global goal. The study was planned and conducted to comparatively evaluate the different criteria which are considered by the software developer during the selection of effective security requirements engineering approach. A survey form was prepared to determine the prioritization of the characteristics incorporated when choosing effective security requirements engineering approach for the development of trustworthy healthcare software system. This form was given to 25 security experts to collect their estimation on the pair-wise criteria comparisons and fuzzy TOPSIS model was created. A set of ISO 27005 standard criteria represents the balanced hierarchical structure consisting of the seven main criteria and five alternatives incorporating the objectives and criteria when choosing the effective alternative as a security requirements engineering approach. Some of the popular SRE approaches which are used in this study are SREP, SQUARE, STORE, MOSRE and SREF which are represented by A1, A2, A3, A4, and A5 respectively. The Fig.\u00a0We mailed pairwise questionnaire form based on seven points scale to each of the security experts. The security experts were given a questionnaire that contained a pairwise comparison sheet. The members consisted of 25 security experts who were serving in the different software organizations and who had experience exceeding 15\u2009years having deep knowledge about security requirements engineering process during software development. All security experts were security domain experts who practice different security requirements in their working places to build quality software products and have valuable knowledge about the different modern threats and software attack mechanism. They responded about their satisfaction with their choices. Further the collected data analyzed separately for each security expert. The following Table\u00a0Many multi-criteria-decision-making (MCDM) methods were developed in order to rank alternatives differently. Although evaluations of alternatives offered by MCDM methods may sometimes be in contract, there are circumstances where distinct MCDM methods produce very different recommendations \u201326. A fiSeveral researches are available wherein the authors used the fuzzy TOPSIS method to develop effective decisions. Ashrafzadeh et al.  presenteij)m\u2009\u00d7\u2009n. Let W\u2009=\u2009 be the weight vector of criteria, satisfying In this method if each characteristic takes on asymptotically raising or lowering variation, then maybe an ideal solution can be easily defined. That solution consists of all possible to achieve best attributes, since the worst solution consists of all attainable worst attribute values. Assumed a decision-making issue with multiple criteria has n alternatives, A1, A2, ..., An and m criteria, C1, C2, ..., Cm. Each alternative is assessed against the criteria of m. All the values/ratings are allocated to alternatives regarding decision matrix represented by X and , e distance between the two can be calculated as follows:d is the distance between two fuzzy numbers, when given two triangular fuzzy numbers  of different alternatives is estimated as 0.438, 0.74, 0.842, 0.322 and 0.341 for A1, A2, A3, A4 and A5 respectively. The findings show the A3 has highly effective and efficient security requirements engineering approach for trustworthy healthcare software development.Today\u2019s several malicious attempts are by technological flaws in healthcare applications such as SQL injection, session hijacking or cross-site scripting. Healthcare applications which are susceptible to such vulnerabilities have become a convenient target for cyber criminals and therefore most frequently lead to confidential information breaches. The confidentiality of patient\u2019s data remains the most significant challenge to reach when contemplating the implementation of Healthcare Information Systems (HIS) in the healthcare sector. Recently, so many studies were performed to recognize potential healthcare security threats, as well as a variety of alternatives were suggested to facilitate security requirements for security-critical healthcare applications. In this study we undertake the powerful fuzzy TOPSIS technique for the selection of effective Security Requirements Engineering approach to assist the software developers in developing trustworthy healthcare software.Based on the findings, it is clear that Security Threat Oriented Requirements Engineering (STORE) methodology is the most effective SRE approach based on the security experts\u2019 selections. This undoubtedly shows that the recognition of threats is very much significant and every security requirement engineering approach should consider this criterion. In many existing security requirements engineering methods, the threat identification has played a significant role in eliciting effective security requirements for a software project. Prior to eliciting the effective security requirements of a healthcare software system, it is significant to identify all the possible threats to the software system. Assessing the threats to a software system assists software developers to build complete and accurate security requirements , 29.Further risk analysis and stakeholder\u2019s involvement in the security requirements engineering process is also important. Although considering stakeholder\u2019s view in security requirement engineering is an important concern, but only some security requirements engineering approaches to address this concern. This doesn\u2019t mean that it is impossible to consider the views of different stakeholders using other methods. However, most of the security requirements do not capture this issue in their various activities \u201333. The After the criteria were prioritized accordingly, the model used in this study enabled us to analyses the main concept of the consistency of preferences made by the security experts. Furthermore, the main purpose of this study is to provide a model enabling security experts to make a more consistent decision for trustworthy healthcare software development. After the features of the security requirements engineering method became clear, this model can be used to predict effective security requirements engineering approach selection in the real world. This study determined the priority of alternatives that are considered in selecting an effective security requirements engineering method respective of criteria for trustworthy healthcare software development. These criteria highlight the prioritized SRE approaches to which a software developer should pay attention.Effective security requirements engineering approach selection decision is essential for the trustworthy healthcare software development. Determining among the many existing SRE methods is a challenging decision-making problem due to the fact that each approach has advantages as well as disadvantages. We implemented the results of a study on the application of fuzzy TOPSIS methodology. A set of ISO 27005 standard criteria identified based on the literature review and organized into a rational hierarchical structure consisting of the seven main criteria and five alternatives. The consistency ratios were less than 0.10 for all the 25 security experts in collected form responses. The research findings suggest that the STORE approach  is more effective than SQUARE (0.74), SREP (0.438), SREF (0.341), and MOSRE (0.322) in manipulating performance towards security requirements engineering approaches. Determining weights of essential motivation, purpose, and consciousness focus areas can help security decision-making and compliance with policy, and support design of effective security requirements engineering. However, these weights may in turn be affected by local organizational and educational factors. The presented fuzzy TOPSIS results in this paper can be used to select or design an effective security requirements engineering approach that may assist the software developers in developing a trustworthy healthcare software system. Several other fuzzy decision-making approaches are available like VIKOR, fuzzy ANP, PROMETHE, and many others can be used for future research, and their findings can be compared with the findings obtained in this study. The outcome discussed in this research may be used by the software professionals working in the clinical, education, and healthcare activities related to software development."}
+{"text": "The high incidence of fungal infections has become a worrisome public health issue, having been aggravated by an increase in host predisposition factors. Despite all the drugs available on the market to treat these diseases, their efficiency is questionable, and their side effects cannot be neglected. Bearing that in mind, it is of upmost importance to synthetize new and innovative carriers for these medicines not only to fight emerging fungal infections but also to avert the increase in drug-resistant strains. Although it has revealed to be a difficult job, new nano-based drug delivery systems and even new cellular targets and compounds with antifungal potential are now being investigated. This article will provide a summary of the state-of-the-art strategies that have been studied in order to improve antifungal therapy and reduce adverse effects of conventional drugs. The bidirectional relationship between Mycology and Nanotechnology will be also explained. Furthermore, the article will focus on new compounds from the marine environment which have a proven antifungal potential and may act as platforms to discover drug-like characteristics, highlighting the challenges of the translation of these natural compounds into the clinical pipeline. Candida, Cryptococcus, Aspergillus, and Pneumocystis species, being the cause of cryptococcosis, candidiasis, aspergillosis, and pneumocystis pneumonia, respectively . The. The4]. There are 20\u201340% mortality rates with invasive mycoses, therefore these figures need to be improved;The increase in patients undergoing prolonged antifungal therapies reflects the need to develop better fungicidal drugs and thus reduce the length of the treatments and the costs associated;There is still space for improvement in pharmacokinetics and pharmacodynamics, in order to reduce the frequency of drug use;More attention needs to be given to the host toxicities and drug\u2013drug interactions of current therapy so that their effects can be eliminated or, at least, minimized;New therapy groups with different mechanisms of action are needed; this way, these new drugs may synergize with present ones and allow better responses;There is an alarming growth in antifungal resistance in all therapeutic groups .Nevertheless, the design and development of new drug delivery systems or even new antifungals is an emerging need, owing to the following facts :There arNanotechnology is an emerging field of science that has shown an undeniable versatility and has boosted a revolution when it comes to medical treatments, quicker diagnosis, cellular regeneration, and drug delivery ,10. The Nanoparticles have been employed in pharmaceutical formulations because of their ability to alter and improve the pharmacokinetic and pharmacodynamic properties of the drugs. This is given to their capability to increase the solubility and stability of the drugs, to allow a controlled release and to exhibit biocompatibility with tissues and cells, which is reflected in an overall improvement on therapeutic efficiency ,12. In aIn However, the efficacy and human safety of these new therapies remain uncertain in most of the articles found in literature. They generally lack controlled clinical trials and sometimes the suggested routes of administration are less practical, or the production cost may hinder the replacement of the conventional treatment. Nevertheless, in other cases, the opposite is verified, and some options have potential to become a viable first line treatment . MoreoveThe chemical features and simultaneous multiple mechanisms used by nitric oxide, chitosan, and metallic nanoparticles make the likelihood of resistance development unviable  ,105;The resistance mechanisms can be prevented by packaging multiple antimicrobial drugs within the same nanoparticle, because the likelihood of multiple simultaneous gene mutations in the same cell is low. The most striking examples are the encapsulation of antifungal drugs in chitosan or silver nanoparticles, combining the antifungal properties of both and decreasing the possibility of drug resistance ,106;Some nanoparticles, such as liposomes and dendrimers, are able to overcome the resistance mechanisms of decreased uptake and increased efflux of drug from the microbial cell. Liposomes are able to quickly fuse with the plasma membrane of the microbial cell and release a high concentration of drug into its plasma membrane or cytoplasm, thereby circumventing the decreased uptake mechanism of resistance. This means a faster delivery and avoidance of the transmembrane pumps that catalyze increased efflux of drugs. Dendrimers, on the other hand, are extensively branched molecules, whose surface can be filled with positively charged quaternary ammonium compounds, which bind to negatively charged microbial cell envelopes and increase membrane permeability. This allows the entrance of more dendrimers to the microbial cell, the flow of its cytoplasmic contents to the exterior, and the ultimate destruction of the microbial cell membrane. This goes to show that dendrimers are also able to surpass the resistance mechanism of decreased uptake of drug . Other nNanoparticles have been used to target antifungal drugs to the specific site of infection, allowing the local release of high concentrations of drug, while keeping the total dose of drug administered low. This high local dose is able to destroy the infecting fungi before they can develop resistance, thereby overcoming this worrisome issue and translating into fewer side effects upon the patient .Despite the uprising of these issues in antifungal therapy, there are several mechanisms by which nanoparticles overcome the development of resistance mechanisms:That being said, it is also important that the research done, not only focuses on formulating these systems, but also in overcoming the major challenges that their placing on the market faces: the physical instability of nanoparticles, their small capacity of drug loading, the cytotoxicity/immunogenicity, and the high cost of production and standardization, given the complexity of the formulations. Besides that, there is almost a complete lack of studies in vivo as reaching the therapeutic range needed to perform these studies has proven to be an arduous job. That lies in the fact that, in many cases, there is an anticipated release of the drug, aggregation and precipitation of the nanoparticles, and the accumulation in non-target tissues.Mycology and Nanotechnology have created a bidirectional relationship throughout the years. This dynamic interface between mycology and nanotechnology led to the creation of the term \u201cmyconanotechnology\u201d  110]. N. N110]. Metallic nanoparticles have been used to eliminate fungi that are pathogenic to Man and to plants, because of their intrinsic antimicrobial activity . The exaNanoparticles undergo dissolution processes thanks to their electrochemical potential . This le+ has high affinity to thiol groups in cysteine of respiratory chain enzymes, therefore it uncouples the synthesis of adenosine triphosphate (ATP). Ag+ also binds to proteins of transport from the respiratory chain, causing the leak out of protons and thus the collapse of the proton motive force. Furthermore, Ag+ obstructs the uptake of phosphate and so promotes the efflux of intracellular phosphate . Th. Th10]. The biological synthesis can either be intracellular or extracellular according to the nanoparticles\u2019 locations. In intracellular synthesis, the ions are transported to the inner part of microbial cells to form nanoparticles in the presence of an enzyme. The nanoparticles formed inside the organism are of shorter size, when compared to the extracellular ones, because there is nucleation of particles inside the organisms. Extracellular synthesis has more applications than intracellular, as there is no need to join cellular components from the cell. The majority of the fungi produce nanoparticles extracellularly as a result of their secretory components that participate in nanoparticles reduction and capping .Both yeasts and filamentous fungi can be used to synthetize nanoparticles. The process of synthetizing filamentous fungi-mediated nanoparticles is easy and cost-effective, since the mycelia in the biomass has high surface area and high intracellular metal absorption. Moreover, the fungi cell wall has many functional groups that facilitate the absorption to the metals .Fusarium solani, a pathogenic fungus isolated from infected onions. Ingle et al. [Silver nanoparticles are the most fundamental among metallic nanoparticles that are involved in biomedical applications, specially cancer diagnosis and antimicrobial therapy . One of e et al.  believe e et al. .Fusarium oxysporum [Cochliobolus lunatus, Beauveria bassiana [Bipolaris maydis [Phoma glomerata, Phoma herbarum, Fusarium semitectum, Trichoderma sp., Phoma glomerata, Phoma herbarum, and Candida albicans [Penicillium brevicompactum, Aspergillus fumigatus, Cladosporium cladosporoides, Chaetomium globosum, and Stachybotrys chartarum [Afterwards, many other scientists were able to optimize the biological production of silver nanoparticles using other fungi, such as xysporum , Cochliobassiana , Bipolars maydis , and manalbicans . Surprishartarum .Candida glabrata and Schizosaccharomyces pombe [Saccharomyces cerevisiae in aerobic conditions [Likewise, yeasts can be useful for nanoparticle synthesis, as they produce enzymes responsible for the reduction of metallic salts and their conversion into elementary nanoparticles. Some examples are the biosynthesis of cadmium nanoparticles by es pombe  and of snditions ,127.The administration of antifungal drugs is not restricted to oral or parenteral routes. Other routes such as transungual, pulmonary, and ocular have also acquired great importance in the treatment of certain fungal infections. The development of a drug delivery system for attaining therapeutic concentration at these target organs is a challenge that requires a comprehensive understanding of the dynamics and specific features of the nails, lungs, and eyes, respectively.Tinea ungium) caused by dermatophytes (such as Trichophyton rubrum) or yeasts are concerned [This special topical route acquires singular importance in antifungal therapy, mainly when onychomycosis  along with an edge activator molecule. This combination causes a rearrangement in the lipid bilayer of transethosomes, allowing them to have both higher deformability and skin permeation/penetration than the other deformable molecules. The formulation of these vesicles also presents the advantage of being quite easy to scale up, which constitutes an enormous advantage for industrial purposes . ContrarAn econazole nitrate loaded transethosome gel for transdermal delivery has been developed. Comparing it with the marketed cream of econazole nitrate, the authors found out that the former presented less ex vivo penetration, higher ex vivo skin retention, and higher in vitro antifungal activity . TransetRegardless of the fact that transethosomes represent the state-of-the-art in ultradeformable vesicles, they still have some drawbacks that are important to mention such as the solubility that the drugs need to fulfil in both lipophilic and aqueous environment so that they can reach the dermal microcirculation and access the systemic circulation. In addition, the molecular size of the drug needs to be reasonable to make the percutaneous absorption possible (40\u2013200 nm) .Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) have become very attractive options to the development of drugs for cutaneous application and as an alternative to overcome the drawbacks of liposomes. They provide an occlusive effect by virtue of their ability to form a surface film that reduces the transepidermal water loss, which can reduce the atopic eczema symptoms and improve the skin appearance . BesidesOne application of SLN and NLC is the incorporation of azoles to treat cutaneous fungal infections. The most common azoles  are extremely water insoluble, being very difficult to administrate and to release those drugs in infection sites. However, compounds with lipophilic character can be efficiently encapsulated in SLNs .In 2010, a formulation was developed in which the SLN nanoparticles were dispersed in a hydrogel to carry miconazole nitrate (MN), an azole frequently used in cutaneous fungal infections . The stuPolymeric nanoparticles are produced either from natural or synthetic polymers, in which the drug is dissolved, trapped, or bound to the nanoparticle matrix. Depending upon the composition of the organic phase and the preparation method, nanocapsules (matrix-like structure) or nanospheres (core-shell morphology) can be obtained ,150.These nanoparticles are capable of carrying proteins, DNA, and drugs, such as antifungals, for cells and specific target organs, which increases the safety profile. Their nanometric size promotes an effective permeation through the cellular membranes and increases their stability in blood flow.It is also important to point out the gelling systems that can be used to formulate polymeric carriers: microsponges and nanosponges, amphiphilic gels and emulgels or gellified emulsions, which play an important role in cutaneous drug delivery .Microsponge and nanosponge systems are polymer-based spheres that can encapsulate or suspend many substances and then be incorporated into a dosage form (hydrogel) or be used for oral delivery ,66,151.A polymeric microsponge based gel system was successfully produced for the topical delivery of fluconazole, with especially great entrapment efficiency, production yield, and extended drug release, which allows a reduction in application frequency, a feature with great importance in recidivist and prolonged fungal infections .A nanosponge based gel formulation loaded with clotrimazole showed controlled release with reduced side effects, indicating the safe and effective profile of these colloidal carriers for topical use . An econAmphiphilic gels are semisolid systems of nonionic nature that can be used as topical/transdermal carriers without promoting irritation of the skin. They aim at delivering the antifungal in a level within the therapeutic window for as long as possible and to avoid fluctuations in plasma drug level .An amphiphilogel of fluconazole was successfully formulated for topical application, presenting overall stability and cumulative drug release within the range expected .Emulgels have dual release control system (emulsion/microemulsion and gel), which increases the overall stability of the antifungal formulations . In compEmulgels are suitable solutions to incorporate hydrophobic drugs in water soluble gel bases, for instance the incorporation of clotrimazole in an emulgel formulation prepared using either Carbopol 934 or HMPC 2910 showed good physical properties, stability, and antifungal activity .Candida albicans equal to the free drug, with the advantage of presenting less toxicity and less mortality associated [A formulation with amphotericin B encapsulated in poly(lactic-co-glycolic acid) PLGA in association with \u03b5-caprolactone was developed and proved the potential of polymeric nanoparticles in preventing cytotoxicity, yet preserving the fungicidal efficacy. With this polymeric combination, the authors described an amphotericin B encapsulation efficacy of 84% and a fungicidal effect for sociated .A nanoformulation of the same drug in PLGA was also developed but, this time, it was functionalized with dimercaptosuccinic acid (DMSA). This acid exhibits tropism to lungs, being appropriate to be included in a formulation in which it is desired a release at this site. In this study an intraperitoneal administration was also tested and verified that the therapeutic effect on paracoccidioidomycosis (PCM) was equivalent to the free drug. The greatest advantage of this formulation was that it only required to be taken every three days, given the slow release of amphotericin B from the nanoparticles, and did not require a daily administration as the currently commercialized formulation .Chitosan is a very versatile polymer owing to the ease presented by their groups to be modified or deacetylated . Some st\u00ae formulation. Moreover, this natural polysaccharide was also employed in the cutaneous release of amphotericin B in wounds caused by infected burns with Candida albicans, promoting better tissue healing and increased antifungal activity, when compared to the conventional formulation [Chitosan nanoparticles containing amphotericin B were initially developed with the aim of avoiding the drug gastrointestinal degradation, increasing the stability and the bioavailability in target organs like the lung, the liver, and the spleen, whilst decreasing renal exposition. The researchers determined that when administered by an oral route, these nanoparticles were effective in treating visceral leishmaniosis, aspergillosis, and candidiasis, showing an efficacy comparable to the parenteral Ambisomemulation .Polymeric micelles present a unique architecture where the hydrophobic core can incorporate hydrophobic drugs, such as antifungals, which leads to a very significant improvement in their aqueous solubility . These sCandida meningoencephalitis [Amphotericin B has been incorporated in polymeric micelles to treat brain fungal infections. The system was intended to improve drug solubility and permeability through biological membranes as well as obviate issues like high toxicity and low efficacy against phalitis .Polymersomes are spherical structures composed of an aqueous core surrounded by a polymeric bilayer membrane, being viewed as synthetic analogues to liposomes. They are very versatile structures, which spontaneously self-assemble from amphiphilic diblock copolymers. This capacity increases drug efficacy and enables them to encapsulate both hydrophilic and hydrophobic drugs, including antifungals ,159. Fur3-PLA as co-polymer. This formulation was compared with two marketed formulations (Fungizone\u00ae and Ambisome\u00ae) in terms of release, molecular organization of amphotericin B, and hemolysis. The results were similar to the marketed formulations, which indicated the potential for further in vivo development [A research group developed an amphotericin B loaded polymersome by solvent injection method, using (PEG)elopment .Dendrimers are homogenous polymeric tridimensional nanoarchitectures characterized by a highly-branched and symmetrical structure consisting of a central core (a single atom or a group of atoms), building blocks of repeating units emanating from the core (generations), and a high density of water-soluble functional groups on the surface  ,162,163.Albeit different types of dendrimers have been described, the most frequently used for antifungal therapy are polyamidoamine (PAMAM) and polypropylene imine (PPI) dendrimers . PAMAM dThree main groups of metallic nanoparticles, gold, silver, and magnetic, can be used for the vectorization of antifungal drugs.Metallic nanoparticles can be synthetized in three different ways: chemically, physically, and biologically. Chemical synthesis has been associated with many side effects related with the absorption of toxic chemical particles on the surface of nanoparticles , thereupGold nanoparticles are used in immunochemical studies to identify protein interactions and in DNA fingerprinting to detect DNA in a sample. They are also used to detect aminoglycosides like streptomycin, gentamicin, and neomycin .Silver nanoparticles are the most efficient on account of their antimicrobial efficacy against bacteria, viruses, and other eukaryotic microorganisms. In fact, they are the most used materials, being applied as antimicrobial agents in textile industry, for water treatment, in solar protectors, etc. .3O4), hematite (\u03b1-Fe2O3), and maghemite (\u03b3-Fe2O3), are the most studied types of magnetic nanoparticles. This type of nanoparticle has received special attention by virtue of their capacity to be influenced by magnetic fields, therefore being easily directed and released in a specific site of the organism [Albeit nanostructures of iron, cobalt, and nickel exhibit superparamagnetic properties and high magnetic susceptibility, superparamagnetic iron oxide nanoparticles such as magnetite ; this is a procedure that is important not only to increase cell internalization and biocompatibility, but also to carry active molecules to the nanoparticle\u2019s surface, essential to drug delivery [Nowadays, nanoparticles of superparamagnetic iron oxide are the ones that answer more efficiently to an external magnetic field, being the ones with more potential to become drug carriers . In ordedelivery .Niosomes are non-phospholipid vesicles made of non-ionic surfactants that serve as drug depots in the body as they release the drug in a sustained fashion through its bilayers. They are also able to improve oral bioavailability of poorly soluble drugs, enhance the skin permeability of topical drugs, protect the enclosed active substance from deleterious factors, and increase the stability of the entrapped drug .Candida albicans with a higher level of drug in vital organs [Nistatin was successfully encapsulated in niosomes and a safe and effective formula for parenteral administration was obtained. This formulation provided reduced nephrotoxicity and hepatoxicity in female Wistar rats and showed pronounced efficacy against l organs . The encl organs . Given tl organs .Ketoconazole niosomal gel showed more prolonged action than conventional ketoconazole formulations, hence it can be developed in order to improve the antifungal activity .Spanlastics are termed as modified niosomes which present better permeability because they have edge activators in their composition, like transfersomes and transethosomes ,145.These systems show great flexibility allowing them to pass through fenestrations smaller than their own radius in order to enter the cell, simultaneously minimizing the possibility of damaging the vesicles while squashing .Firstly, there were ketoconazole-loaded spanlastics for ocular drug delivery  and thenMicroemulsions are colloidal carriers that consist of a liquid dispersion of oil and water stabilized by an interfacial film of surfactant. Due to this, they are able to incorporate drugs of different lipophilicity . They arWhen it comes to drug delivery through stratum corneum, microemulsions are very resourceful considering the ability of the oils and surfactants included in their composition to act as skin penetration enhancers ,172. FurCandida albicans, along with better drug skin penetration [Terbinafine hydrochloride is an example of a slightly water-soluble drug, which presented higher solubility, an increase in the dissolution rate, and better efficacy when incorporated within a microemulsion ,171. Liketration .Nanoemulsions are thermodynamically and kinetically more stable than emulsions, so they have been evaluated as colloidal carriers to improve the efficacy and tolerability of some antifungal drugs. The capacity of nanoemulsions to dissolve large quantities of drugs with low solubility, their compatibility, their ability to protect drugs from enzymatic degradation and hydrolysis as well as their capacity to penetrate the deeper skin layers, turns them into ideal drug delivery vectors ,174.A nystatin nanoemulsion for topical application was developed and was found to have a higher antifungal effect than nystatin itself, representing a therapeutic improvement .Silicon dioxide nanoparticles have been studied as drug carriers to enhance the antimicrobial activity of some drugs, because of their biodegradability, low toxicity, capacity to stimulate macrophages, and the ease by which they are synthetized and modified. The foremost advantage of these nanoparticles is that they can be loaded with large amounts of drug ,175.Nitric oxide-silica nanoparticles with proven anti-biofilm activity ;Metal modified silica nanoparticles, which can include silver or copper, metals that have a very well documented antimicrobial effect, derived from the cell membrane and DNA damages, interaction with enzymes from thiol groups or are associated with generating hydrogen peroxide ;Surface-modified silica nanoparticles by quaternary ammonium compounds loaded with antifungal agents ,178;Bioglasses and bioceramics .There are four main types of silica nanoparticles, but their antifungal potential is yet to be studied in some cases:Amongst them, mesoporous silica nanoparticles are the ones who have become the most promising candidates for many biomedical applications, specifically for antifungal therapy, because of their uniformed mesoporous tunnels and narrow pore size distribution. Besides that, they present outstanding biocompatibility and chemical stability and can be degraded and metabolized over a relatively short term. These aspects allow high drug loading and reduce the probability of particle-induced toxicity ,180.Candida albicans was treated with silica nanoparticles functionalized with amphotericin B and the other group was treated with officinal amphotericin B. It was possible to observe that the first group showed a significant increase in survival rates [Candida albicans than colloidal silver [In a study, one group of mice with candidiasis induced by al rates . This col silver ,178.In order to increase the oral bioavailability of itraconazole, a poorly water-soluble antifungal drug, mesoporous silica particles formulation was developed. It was concluded that ordered mesoporous silica can, in fact, be considered a promising carrier seeing that it enhances the oral bioavailability of extremely low water-soluble drugs .It has been demonstrated that metals can easily accumulate in soil and then enter the food chain, thus the combination of Ag+ ions with silica prevents the formation of these ions and reduces its toxicity . That isFrom 1981 to 2014, 32 new chemical entities were placed on the market to treat fungal infections: one from a biological source (interferon gamma-n1), that is a peptide produced by a biotechnological procedure; three derived from a natural product that suffered a semisynthetic modification ; 25 totally synthetic discovered by random screening or through the modification of an existing agent  .In fact, almost 90% of the antifungal agents approved within this period were from a synthetic origin. This paucity of natural products in modern treatments remains a reality and 1950s agents like amphotericin and griseofulvin are still widely used .Mentha pulegium L., commonly known as pennyroyal, a flowering herb with antitussive, carminative, and antiseptic effects. This herb was successfully used to synthesize stable colloidal silver nanoparticles with promising antifungal effects against Candida albicans [One good example of an efficient employment of natural products to nanoparticle synthesis happened with albicans .On the other hand, the marine environment represents a valuable and unexplored platform to the discovery of new compounds . In TablSponges have always interested pharmacologists, chemists, and biologists as a rich source of antimicrobial compounds with peculiar activities. These colonial organisms have a sessile nature, hence the necessity of producing compounds as a way of protecting themselves, communicating, or modulating their cellular functions .Peptides have high relevance as potential drugs, given their large spectrum of bioactivity. Sponges that belong to the Theonella gender are a recognized source of uncommon peptides with antibacterial, antifungal, and anti-HIV properties. The antifungal activity of some of these peptides is even surplus to other commercial formulations, as seen through their diffusion zone in the agar diffusion method .Although some formulations containing marine compounds are already being subjected to clinical trials, none of them target fungal infections .Theonella swinhoei from Papua New Guinea [Candida albicans wild strains and also strains resistant to amphotericin B [Theopapuamide A is a cyclic depsipeptide (a peptide in which one or more amides were replaced by an ester group) being firstly isolated from the sponge w Guinea . Its isow Guinea . It is cericin B . In addiericin B ,184.Curcuma zedoaria, Psidium guajava, Plectranthus amboinicus, Lippia alba) have shown activity against Candida spp. and others  against filamentous fungi. Despite the streamline nature of the discovery process, the biological activity found during the screening of plant extracts, may not be experimentally reproducible. This can be due to many reasons: the chemical constituents in the crude extracts may be different, the solvent used to extract may destroy some compounds and chemical composition may vary according to the growth stage or geographic origin. Some components of essential oils, mainly terpenes or terpenoids, such as eugenol, camphor, curcumin, geraniol, and linalol show activity against a wide variety of Candida species and others like clemateol or citral are active against Trichophyton spp. or Malassezia spp. respectively. Propolis, which represents the resinous substances collected from plants by bees, demonstrated activity against Candida spp., some dermatophytes, and onychomycosis. Ajoene, a compound derived from garlic, has shown effectiveness in the treatment of paracoccidioidomycosis and Fusarium spp. infections. Other compounds such as saponins, alkaloids, flavonoids, coumarins, xanthones, lignans, and tannins also presented antifungal activity [Besides the marine environment, a considerable number of studies have been conducted on medicinal plants and alternative compounds, such as secondary metabolites, phenolic compounds, essential oils, and extracts. Some plant extracts  range from rather strict to non-existent and vary between regions, being under the surveillance of different authorities .\u00ae , Abelcet\u00ae  and Ambisome\u00ae , all lipid-based formulations [\u00ae) is also available on the market [Several pharmaceutical industries and authors have been performing clinical trials on antifungal nanomedicines. Despite the growing improvement on applying nanotechnology for drug development, just a few of those nanomedicines have been approved for their clinical applications. The major issues with clinical trials are their time-consuming process and the difficulty while accessing and researching information on previous nanomedicine trends. In the mycology field, amphotericin-B is the active substance that represents the majority of the nanomedicines already available in the market: Amphoteculations ,192. An e market . In TablThe present pace of antifungal drug development is highly unlikely to keep up with the clinical needs, especially with the uprising of resistance to current agents. Therefore, more therapeutic answers to fungal illnesses are urgently needed.The subject of sustainability and environmental impact has gained considerable relevance in today\u2019s societies and scientific research, motivating a heated debate on whether there should be an active research for new natural compounds with biological activity or if the pillar of today\u2019s research for new compounds should be high throughput screening. The fact is that two out of the three major classes of antifungal drugs (polyenes and echinocandins) were screened from natural products. The utilization of natural compounds, such as chitosan is, irrefutably, a more sustainable option and brings both environmental and pharmacokinetic advantages to nanotechnological approaches. Compounds from a marine origin exhibit an undeniable potential, but their activity and toxicity mechanisms are yet to be clarified.Nanoparticles have been presented as promising solutions, mainly due to their ability to target specific sites where fungi are harbored, their capacity to enhance the pharmacological effect of drugs, optimizing their physiochemical characteristics, thereby allowing an administration through a more comfortable route. All these features can enable lower dosing, more comfortable regimens, increased bioavailability, and less serious adverse effects.Special attention has been given to certain kinds of nanoparticles nowadays mainly because of the outstanding features they exhibit: (a) magnetic nanoparticles and their capacity to directly restrict fungal growth, (b) ultradeformable vesicles (transethosomes) and their ease on scaling up, (c) mesoporous silica nanoparticles and their high drug loading, (d) polymersomes and their ability to carry both hydrophobic and hydrophilic substances and to respond to external stimuli, (e) PAMAM dendrimers and their versatile and biocompatible structure. We speculate that future antifungal therapies will mostly lie in these five types of nanoparticles and will take advantage of the current knowledge of using them for other purposes .These new nanotechnological systems should be able to surpass the issues already mentioned and should also mean a significant upgrade when comparing to the conventional treatments, fighting antifungal resistance, presenting a broad spectrum of activity, with an emphasis on potency increase and little host toxicity, thus having the potential to be industrially produced.Interdisciplinary cooperation may be the key to a striving success of nanomedicine and nanotechnology, not to mention a proper exploitation of natural medicinal products. Physicists, health-care researchers, and clinical researchers have complementary knowledge that could be put together  so that more effective, practical, and safe nanoparticles are designed. That would increase the chances of an industry funding and, ultimately, a rethinking of current antifungal arsenal, while providing a better quality of life for patients."}
+{"text": "Background: To assess the conduct of delivery of public health services at the municipal level in Denmark by applying services enlisted in the Essential Public Health Operation framework (EPHO) of WHO. Methods: We conducted individual qualitative interviews with key informants working with public health using a self-assessment survey tool in order to obtain an in-depth understanding of the interrelation or multidisciplinary work in Kolding Municipality. The developed self-assessment survey tool entailed questions about essential public health activities performed in a municipality. Results: The Municipality organizes and contributes to core service delivery EPHOs, namely health protection, health promotion, and disease prevention. It collaborates with the general practitioners and the Region of Southern Denmark, responsible for hospital care, to fulfill the selected EPHOs. Conclusions: To obtain a comprehensive picture of the organizations that deliver public health services within a municipality, it is necessary to conduct interviews with representatives from those organizations as well. Additionally, the results from this study can be used to improve the survey tool further and hereafter conduct a nationwide survey in Denmark, as well as other European countries. The public health discipline has in the last 50 years developed rapidly and contributed significantly to the improvement in population health . The risThe European Health 2020 Policy tackles the aforementioned challenges, presenting new rationally goal-oriented public health strategies for promoting health . The MemVery few studies have analyzed or described how public health systems are organized, financed, and evaluated in different European countries. Allin et al.  describehttps://www.euro.who.int/__data/assets/pdf_file/0004/160519/e96442.pdf?ua=1) is available in In Denmark, the Danish Health Act 2005 (Sundhedsloven)  delegateTo develop a survey tool that fits the Danish system and apply it in Kolding Municipality.To identify who is providing core service delivery EPHOs at the municipal level in Denmark.The primary aim of this pilot study was to assess organizational systems and the coordination of delivery of three core EPHOs  in a Danish municipality by applying criteria and services enlisted in the EPHO framework of WHO. We selected Kolding Municipality to study the organization of public health services. Kolding is located on the Eastern part of the Jutland peninsula and, with a population of about 93,175 , it is one of the biggest Danish municipalities; the core city of Kolding has about 61,121 inhabitants. Kolding Municipality was selected due to its size and convenience in regard to access, location, and time. The specific objectives of the present study were:To obtain an in-depth understanding of the interrelation or multidisciplinary work in Kolding Municipality, it was optimal to conduct individual qualitative interviews with key informants working with public health using a self-assessment survey tool . This foA self-assessment survey tool based on the selected EPHOs  was developed; it is based on a set of 20 Indicators of Local Health System Performance developed by Mays . The guiThe survey consisted of 17 questions, which sufficiently covered the important aspects of public health activities in local communities in Denmark. The survey began with a brief profile description of the participant, which included their contact information, profession, the unit they worked in, and information about the duration of their employment in Kolding Municipality. This is followed by the organizational questions asking the participants whether a specific activity was performed in their jurisdiction. We translated the survey into Danish.Two individual interviews were conducted: One with a health consultant within the Health and Training unit and the other with the Chief of Health and Training unit (senior administrative). However, conducting the survey in an interview format allowed us to collect in-depth data information and gave us feedback on what needed to be revised in the survey tool such as: Amending ambiguous phrasings and terms; tailoring the questions according to a health or non-health unit; not asking questions about law and regulations, because the responsibility lies within the government.A copy of the survey tool was sent to a health consultant within Kolding Municipality to identify key participants accordingly. Ideal participants were senior administrators within the health unit who had general knowledge about the types of public health activities, performed by their agencies and by other organizations within the community.In collaboration with the health consultant, four senior administrators and one health consultant were identified within the Health and Training unit and invited for individual interviews. The health consultant accepted the invitation and worked in the field of alcohol and rehabilitation. Out of the 4 senior administrators, only one accepted the invitation on behalf of all of them. It was particularly challenging to access informants. One key reason for this was the lack of time and resources on their side. However, the two informants were viable as we obtained in-depth information about the essential public health activities (EPHOs) performed by Kolding Municipality. The senior administrator was the chief of the Health and Training unit. The tasks of the Chief of Health and Training are to develop, coordinate, and implement health promotion initiatives, including creating frameworks for equal access to health through development and interdisciplinary cooperation in the areas of healthy food, smoking, alcohol, exercise, and mental health.Preliminary exploration of the data by reading through the transcript and writing memos;Sorting the data based on the EPHOs  and their sub-operations .Steps in the qualitative analysis included:The following section presents the findings on how Kolding Municipality organizes and contributes to core service delivery EPHOs, as well as the contribution and participation of other agencies or health organizations. The findings showed that Kolding municipality is responsible for part of the services described in the core EPHOs only.Within Kolding Municipality, the Health and Training unit is mainly responsible for providing health services in the areas of nutrition, smoking, alcohol, physical activity, and mental health. Within chronic diseases, prevention efforts are aimed at addressing diabetes, cardiovascular diseases (CVD), osteoporosis, and chronic obstructive pulmonary disease (COPD). The municipality is not responsible for public health services such as food protection, occupational health protection, immunization programs, and screenings programs. This is aligned with the Danish Health Act 2005 (Sundhedsloven) , which oIn the following, we present services where Kolding Municipality has full or partial responsibility for provision of core EPHOs.Within Kolding Municipality, the City and Development Department is responsible for environmental health including water, soil, housing, and occupational health, and has an environmental policy framework, and planning and housing guidelines. The policy framework and guidelines are developed based on the established directives from the Food and Environmental Protection Agency. However, the Health and Training unit is not responsible for environmental health.The Health and Training unit offers workplace health programs to public and private corporations. The aim is to promote and support healthy choices and hereby create an enabling environment for healthy behaviors among workers. However, the Health and Training unit is not responsible for the legislative framework for occupational health.Occupational health services in Denmark are primarily regulated by the National Working Environment Authority.The municipality establishes their health policy and health plan based on the overall guideline provided by the Ministry of Health. It is the Association of municipalities (KL) and Association of Danish Regions (Danske Regioner) who specify and apply the national guidelines.Kolding Municipality is also part of Sundhedsstrategisk Forum , which is a meeting forum for all the municipalities in the Region of Southern Denmark. It is a platform where representatives from the municipalities, hospitals, and general practitioners (GPs) meet regularly to support, discuss, and formulate public health strategies. The forum addresses key health issues such as cancer, diabetes, cardiovascular diseases, musculoskeletal disorders, and mental health, and establishes health plans that describe the responsibilities of the municipalities, hospitals, and GPs in the specific health areas.The municipality also has a communication network with the National Health Board, the National Serum Institute (SSI), and the National Institute of Public Health (SIF).The Health and Training unit develops, coordinates, and implements health promotion, and coordinates efforts carried out by different teams. They ensure that the citizens have equal access to healthcare through interdisciplinary cooperation in the areas of nutrition, smoking, alcohol, exercise, and mental health. Within chronic diseases, prevention efforts are aimed at diabetes, CVD, osteoporosis, and COPD. The municipality collaborates with the Region of Southern Denmark and the Danish Regions and conducts a national health profile survey \u201cHvordan har du det? (How are you?),\u201d which looks at contributing factors ) to prioritize health needs.In addition, Kolding Municipality participates in a nationwide youth profile survey conducted by Senior- and Social Department and Police (SSP-Network). It provides a descriptive profile of changes in the youth development related to health, crime, drugs, education, and working or leisure time.Moreover, the municipality has timely communication with GPs regarding health promotion and prevention programs including counseling and support programs to patients at the healthcare center. Likewise, Kolding Municipality cooperates with the hospitals about care and rehabilitation that do not take place during hospitalizations.The Health and Training unit of Kolding municipality coordinates intersectoral health promotion and prevention activities as well. They engage in health promotion programs with, e.g., Senior Unit, Job Center Kolding, and Abuse Center. Every department or team has various collaborations (intern or extern) with different organizations when working on certain projects. Central to these partnerships are that the organizations are either directly or indirectly tied together.The main responsibility of primary prevention rests with the GPs, the hospitals, and the SSI. The SSI is responsible for surveillance and control of communicable diseases, while the GPs and hospitals are obliged to report instances of certain communicable diseases to the SSI. The GPs delivers public health services related to vaccination and immunization programs. The municipality including Health and Training is not responsible for primary prevention programs, and the responsibilities lie within another jurisdiction.The primary care sector, hospitals, is mainly responsible for secondary prevention services such as screening programs for early detection of diseases and maternal child health programs. However, Kolding Municipality delivers maternal and child health services. It includes educational and management programs for first-time mothers, post-delivery, and infants care programs.It is Kolding Municipality\u2019s responsibility to provide rehabilitation and chronic pain management programs to patients before and after hospitalization. Furthermore, the municipality oversees nursing homes and provides care at homes for the elderly. The senior department is mainly responsible for elderly care. Support and educational programs for marginalized and vulnerable groups are present.The findings from the survey showed that Kolding Municipality is only partially responsible for services described under three core EPHOs, which is fully in line with \u00a7 119 of Danish Health Act, nr. 546 from 24 June 2005 . The munA major challenge with such a mapping exercise is to construct a reliable and comprehensive mapping/survey tool. The WHO Self-assessment tool  containsAnother important issue is selection of participants; in informal discussions with participants, it was mentioned that the survey tool requires quite a comprehensive knowledge of public health and EPHOs as such. Therefore, a high level of expertise is needed to complete the survey. This knowledge criterion would gain even more importance if the survey addresses non-health departments as well. Due to the intersectorality approach, inclusions of, for example, environmental, or social affair units would be highly requirable in future work.Additionally, the participants  were unaware of the EPHOs, which posed as a barrier. Recent findings from the midterm report on the EAP-PHS also highlight that many key public health actors do not have knowledge of the EPHOs, which makes the assessment and implementation of the EPHOs challenging indeed .Despite all limitations, this was the first pilot study that investigated how a local public health organization in Denmark organized and contributed to core service delivery EPHOs, as well as the contribution and participation of other agencies or health organizations. A strength of the present study was that the survey was conducted in face-to-face interviews, which enabled us to collect in-depth information. This also allowed the interviewer to clarify questions that the participant found confusing or ambiguous and, therefore, provided feedback on what needed to be improved in the self-assessment survey tool.This pilot study can be used as a framework to conduct a nationwide survey in Denmark related to core service delivery EPHOs. The findings from this study can be used to refine the survey tool so it is even more specific to the Danish system. As many of the sub-operations in the core service delivery EPHOs belong to non-health units, it is necessary to include those units in the future to obtain a more comprehensive picture of how delivery of services is organized. The \u201cideal respondent\u201d is a senior administrative who has in-depth knowledge of the types of public health activities performed by their municipality and by other agencies within the community. To enhance clarity in organization of public health systems responsible for the conduct of EPHOs, similar surveys would be recommended to other countries.In the present study, a survey tool, based on the set of 20 Indicators of Local Health System Performance by Glen P. Mays and the guiding criteria laid in the EPHO self-assessment tool, was developed. The core function of the survey instrument was to assess the essential public health activities performed within a municipality in Demark and the types of organization or key actors that contributed to these activities. The developed survey tool was tested using Kolding Municipality, the Health and Training unit, as a setting. The findings showed that Kolding Municipality organizes and contributes to core service delivery EPHOs at limited extent and in cooperation with internal sectors and other health and non-health organizations. Due to the existence and use of the WHO public health service self-assessment tool, it was not the aim of our study to assess the quality of provision of public health services . DespiteMore research is needed in the area of public health system research in order to draw a comprehensive picture of delivery of public health services within a municipal level in Denmark and internationally. A detailed description of provision of individual public health services in terms of organizations should allow for better harmonization and cooperation among different units or institutions involved in the work. Although other national health systems might need different tools related to the issues of system governance, going in depth in EPHO 6 was not objective of this paper, the pilot study can be useful for assessing the delivery and coordination of public health services within other Danish-relevant governance settings.The findings from this pilot study can be useful for assessing the delivery and coordination of public health services within other relevant governance settings ,17."}
+{"text": "MicroMED ) instrument was selected for the ExoMars 2020 mission to study the airborne dust on the red planet through in situ measurements of the size distribution and concentration. This characterization has never been done before and would have a strong impact on the understanding of Martian climate and Aeolian processes on Mars. The MicroMED is an optical particle counter that exploits the measured intensity of light scattered by dust particles when crossing a collimated laser beam. The measurement technique is well established for laboratory and ground applications but in order to be mounted on the Dust Suite payload within the framework of ExoMars 2020 mission, the instrument must be compatible with harsh mechanical and thermal environments and the tight mass budget of the mission payload. This work summarizes the thermo-mechanical design of the instrument, the manufacturing of the flight model and its successful qualification in expected thermal and mechanical environments. MicroMED  was developed by a consortium of Italian research institutes and Spain\u2019s space agency on the basis of the heritage of MEDUSA , a particle counter designed for the Humboldt payload ,2. MicroMicroMED will be mounted on the Dust Suite of the surface platform onboard the ExoMars 2020 ESA and Roscosmos mission. The Dust Suite payload is composed of five sensors developed to image the landing site, monitor the long-term climate, and perform investigation and studies of the Aeolian processes on Mars. The instrument working principle is typical of optical particle counters ; from the light profile levels and duration information about the dust grain size and speed can be obtained.In order to be approved for launch with the mission, the instrument must prove compatibility with harsh mechanical and thermal environments, due mainly to the launcher and the descent module dynamic excitations foreseen in the mission profile and predicted temperature scenarios on Mars. Moreover, a limited mass allocation of 500 g including electronics was made available for this instrument. The feasibility design of the MicroMED has already been reported in previous studies ,11,12,13In this work, the detailed thermo-mechanical final design of the instrument is reported along with the experimental activity carried out in expected mechanical and thermal environmental conditions to validate the developed numerical models of the instrument. Moreover, the tests required to qualify the MicroMED Flight Model prior to integration on the ExoMars landing platform are presented.MicroMED layout is made by different subassemblies. View of the main parts and components is provided in 2 and compatibility with a low pressure environment. One of the two detectors is used to measure scattered light from analyzed particles, whereas the second one  tracks potential degradations of the laser and of the main detector as the consequence of aging and radiations. More details about the instrument\u2019s optical layout and performances are found in [The optical head (OH) is the main part of the instrument that holds science and reference detectors, the optical collimator (OC), the instrument fluid dynamic subsystem (inlet and outlet parts), and differential pressure sensors and temperature sensors. The selected detectors are Si PIN photodiodes (S5106 type @Hamamatsu), providing a sensing area of 5 \u00d7 5 mmfound in .The selected pressure sensor is a differential MEMS pressure from Amphenol Nova Sensors (NPH-8-002.5-GH MEMS type). Its objective is to measure the differential pressure between the OH and the environment since, combining the measured pressure difference with the pressure loss characteristic curve of the instrument fluidic system, the measurement of the flowrate can be performed, allowing the assessment of the atmospheric dust density . The pressure sensor is an off-shelf component, designed to operate in Earth\u2019s environment, but to be adopted for the mission, it underwent full performance characterization in the expected temperature range, in low pressure conditions, and with the expected dose of radiation during the mission. The performed characterization proved the suitability of the selected sensor for the intended application .The pumping system (PS) is based on a space-designed rotary vane pump with an embedded brushless motor; it must provide the differential pressure required for the atmospheric gas and particles to flow through instrument sampling volume. Design feasibility of the PS concept was already reported by Scaccabarozzi and colleagues , but its3 where the scattering of the particles occurs. The scattered light is focused by a parabolic mirror on the science photodiode. Thanks to the optical and CFD design, MicroMED is able to directly measure both the size distribution and concentration of dust grains suspended in the Martian atmosphere, exploring the particle size range from 0.4 \u03bcm to 20 \u03bcm.The OC concentrates laser light, conveyed by an optic fiber in the center of the OH, generating a sampling volume of 1 \u00d7 1 \u00d7 0.24 mmPt1000 temperature sensors are installed on the sensitive components of the MicroMED  and on the electronics as well, in order to monitor the temperature during operative and non-operative phases and assure safety of the installed components. Moreover, one temperature sensor is located on the tip of the instrument\u2019 inlet, to provide an estimate of the Martian atmosphere temperature. The latter is expected to be different from the MicroMED interface one, and its knowledge is fundamental to accurately evaluate and control the flowrate within the instrument\u2019s fluid dynamic circuit. The control of flowrate is needed, especially to achieve the required dust speed on the sampling volume where science measurements are performed.The main instrument components are mounted on the optical bench (OB), which is the interface with the Dust Suite platform. Finally, the external box (EB) holds the electronics group, mechanically shielding the instrument\u2019s components from external dust or contamination. The electronics allows conditioning and controlling of the subsystems and sensors and acquisition of the instrument housekeeping. The electronics is mounted on the instrument top and the EB provides an EMI shielding.Thermo-mechanical design requirements have been derived from the Surface Platform Information Package document . The exp2. Moreover, size and mass allocations for MicroMED development were limited to a maximum envelope of 148 \u00d7 200 \u00d7 70 mm3 and 500 g mass, respectively.Based on the launch and landing levels and gained experience from previous studies ,20,21,22Temperatures at the Dust Suite Platform interface are between \u221250\u00b0 C to 40 \u00b0C or between \u221220 \u00b0C and 40 \u00b0C for the storage and working phases, respectively. It should be noted that the MicroMED requires sampling of the Martian atmosphere through the inlet. Thus, the thermal design must account for the Martian atmosphere temperature variation and it has to be verified that the instrument does not cause large heat dissipation to the environment through its sampling inlet. Moreover, in order to ease the unit thermal control, the MicroMED is covered with thermal insulator (about 9 cm thickness) with protrusion of the inlet in the Martian atmosphere of at least 1 cm. The expected Martian temperature profiles in hot and cold working scenarios  are showThe temperature trends shown in In order to complete the mechanical design of the MicroMED Flight Model, the finite element (FE) approach is used. Feasibility design of the MicroMED was already assessed in a previous study , showingDifferent FE models of the MicroMED assembly were developed and related analyses were performed to verify the mechanical resistance vs. quasi-static loading and instrument dynamic behavior. The assembly FE model comprises 59,885 tetrahedral and triangular elements and 17,274 nodes. The main subassemblies  and other components  were modeled as lumped masses. This was mandatory in order to manage the FE model without jeopardizing the results\u2019 accuracy. The lumped masses were connected to their supporting structures by weighted links and the OB was fixed simulating the bolt\u2019s connection. The main material for the mechanical structures was aluminum alloy Al7075-T6, whereas the inlet tip, which is directly immersed in the Martian atmosphere, was designed with Vespel SP22. The latter is a well-known plastic material with space heritage. Details about the materials used in the FE model are given in In order to evaluate mechanical resistance in the expected temperature environment, a FE model was developed to investigate instrument behavior for the worst-case temperature scenario . The model comprises the same features previously described with the addition of an aluminum plate 6061, to simulate Dust Suite mounting interface.Results of first three modes of vibration and Von Mises stress along three main axes with quasi-static loading are provided in c\u03c3 is the computed Von Mises stress, lim\u03c3 is the maximum admissible stress, and lim\u03b7 is the related safety factor with respect to the limit condition. As for Equation (1), the MOS shall be positive in order to validate the mechanical design.2K) and 2 W/(m2K) for the hot and cold cases, respectively. Infrared emissivity of the developed model is shown in A thermal model of the MicroMED was developed to determine the temperature ranges in various conditions for the main instrument parts. The unit was therefore modeled with shells representing the main structures, electronics board, and power sources . A convective link with the only exposed surface  was set with a convective coefficient of 0.2 W/ for the hot case and 126 s for the cold case. Power budgets for the PS and for the LG in both the operative cold and hot cases are 2.5 W and 0.4 W, respectively. Cycle time for both the LG and PS is set to 100 s.The applied conditions for environmental temperatures are shown in Computed power exchanges at the Dust Suite interface are 1.7 W and 2.9 W in hot and cold operative scenarios, respectively.Performed FE analyses allowed verification of the MicroMED resistance within expected mechanical and thermal environments. It should be noted that the MOS for static and thermo-elastic simulations is larger than the minimum required. The modal analyses showed that MicroMED dynamic behavior is compliant to design requirements, highlighting first resonance about 60% over the minimum admissible value by design, providing a large safety margin with respect to the expected increased compliance due to real mounting and fixation.The thermal analyses showed compatibility between the requirements for MicroMED\u2019s components and the predicted temperatures. The laser was the only part whose predicted operating temperature exceeded the manufacturer-specified maximum temperature; this result was unavoidable because the max operating temperature for the mounting interface is coincident with the laser operating one. Nevertheless, the predicted laser temperature increase was below 1 \u00b0C, a value that is considered not relevant for the reduction of the component lifetime. Moreover, refined thermal analyses with a more detailed geometrical model of the instrument provide evidence that temperature uniformity is generally achieved on the OH, with maximum temperature difference of about 0.3 \u00b0C for both operational cases.Finally, the temperature of the MicroMED inlet tip is always different from the atmospheric one because of both the low convective exchange with the Martian atmosphere and the limited thermal insulation between the sensor and instrument body. Provided that the inlet tip temperature is the closest approximation of the atmospheric one, a correction procedure shall be developed in case the atmospheric temperature cannot be retrieved from other instruments of the atmospheric package sampling volume.MicroMED main components were manufactured and checked before instrument integration. Main tasks were focused on geometry, mass, design tolerances, and interference checking. After manufacturing, coating (SurTec 650 Tri-chrome) was applied on all the mechanical parts. This finishing is required for any flight mechanics to avoid passivation and stress corrosion of the sensitive 7075T6 alloy.In Developed FE models were validated by means of experimental modal analyses on the manufactured main components.three piezoelectric accelerometers  placed on the top of the MicroMED Flight Model to sense acceleration along three testing directions ; andfour temperature sensors  were used: one was mounted on the MicroMED mounting interface (identified as the temperature reference point (TRP)), and the other three were positioned on the cover, on the OH, and on the inlet in order to have temperature distribution over the instrument during thermo-vacuum phases.In order to perform qualification testing on the MicroMED Flight Model, a protection box was designed and manufactured. The box provides the mechanical and thermal interfaces for the tested item, assuring fulfillment of the planetary protection requirements, even when the MicroMED must be transported and mounted on the Dust Suite interface. Thus, the MicroMED was integrated in a clean environment at the INAF-OAC  clean room and mounted inside the box prior to qualification. In order to measure acceleration levels and temperature during the mechanical and thermal tests, different sensors were applied:Views of the sensors positioning for mechanical and thermal tests and of the instrument during integration are shown in Stability of instrument\u2019s parameters, like the voltage of the reference detector and the PS rotational speed and power consumption, were identified as success criteria for the qualification testing.2, and nominal bandwidth up to 2250 Hz. The test procedure required testing of the unit along the three axes X, Y and Z, identified in accordance with the MicroMED mounting on the Dust Suite, with the acceleration levels and profiles summarized in 2) from 20 Hz to 2000 Hz. This test measures, by means of installed accelerometers, the instrument resonances and tracks any change in the MicroMED dynamic behavior. In order to control applied acceleration at the mounting interface and monitor box accelerations, a triaxial  and two monoaxial  accelerometers were used. Accelerometers were calibrated with Br\u00fcel & Kjaer Calibration Exciter, type 4294. Facility for mechanical testing is based on an LDS V830 SPA-16 electro-dynamic shaker, with LMS SCADAS III controller for sine, random, and shock excitation. The shaker provides 8.9 kN peak force, maximum sine acceleration of 734.5 m/s\u22124 Pa , a CC2C Varian conditioning unit, and connection with 37972A Keysight conditioning unit for temperature reading. Varian DS 402 rotary vane pump and T-Station 85 turbomolecular pumping station were used to achieve the required low pressure level in the vacuum chamber. In addition to temperature sensors on MicroMED parts (read by the instrument electronics) and the four ones installed on the MicroMED during integration on the protection box, additional sensors (named from T5 to T10) were installed on the testing unit and cold interface (T11) of the chamber. Cylindrical thermo-vacuum (TV) chamber (300 mm in diameter and 400 mm in length) was used for qualification testing. Both ends of the cylinder are removable flanges and inside the chamber a LiN2 cooled plate is used as a heat sink. Film heaters were used for fine thermal control. The TV chamber comprises a pressure gauge in the range from 1 bar to 10Qualification requires temperature stabilization at defined test points (variation of less than 1 \u00b0C per hour) and its maintenance for at least 2 h dwell time. Thermo-vacuum levels and cycles for qualification are summarized in Some check points were defined during vacuum testing (at safe temperatures for the instrument\u2019s components) to monitor performance of the MicroMED . Measured temperatures at the TRP are provided in Qualification in mechanical environment of the MicroMED Flight Model was successfully completed. Mechanical testing evidenced survival of the instrument under expected mechanical loads. Functional tests performed after each direction did not show deviations from the nominal behavior of the instrument providing indirect proof that no mechanical changes occurred during the testing. Moreover, changes in measured resonances before and after power vibration tests (sine and random) were found to be within the allowed limits  justified by normal settlement of the mechanical structure bolted junctions.Qualification in the thermal environment was successfully performed. No criticalities were detected during testing as shown by temperature plots in The main result of the successful qualification was that MicroMED Flight Model was ready to be delivered to the Russian team of IKI (Space Research Institute of the Russian Academy of Sciences) for the integration activity on the Dust Suite package.This work provides validation of the thermo-mechanical design of MicroMED, a miniaturized particle analyzer for the ExoMars 2020. Modal and quasi-static analyses performed with finite element model allowed verification of the dynamic behavior and mechanical strength of the instrument, required to be compliant with the expected mechanical and thermal scenarios. The thermo-elastic and thermal analyses provided the generated stresses and the expected temperature distribution in different operational and non-operational conditions, showing that no criticalities were foreseen for the most sensitive components . Moreover, predicted thermo-elastic behavior demonstrated no critical conditions within the qualification temperature range, spanning from \u221250 \u00b0C to 60 \u00b0C. The MicroMED Proto-Flight Model was manufactured and integrated for qualification testing. Qualification campaign was successfully completed both in mechanical and thermal environments, proving MicroMED\u2019s compliance with all conditions throughout all mission phases. Moreover, thanks to the stability of MicroMED\u2019s measured performances during the qualification, compliance with the operative environments of the sensor\u2019s optical and thermo-mechanical designs was demonstrated as well."}
+{"text": "Toxins maintains its standards for the high quality of its published papers. Thanks to the cooperation of our reviewers, in 2020, the median time to first decision was 15.8 days and the median time to publication was 2.8 days. Among the reviewers who reviewed for Toxins in 2020, the following two have been selected to receive the \u201cToxins 2020 Outstanding Reviewer Awards\u201d for the timeliness and quality of their review reports in 2020:Peer review is the driving force of journal development, and reviewers are gatekeepers who ensure that Antonets, Kirill S. from All-Russia Research Institute for Agricultural Microbiology (ARRIAM) and St. Petersburg State University, RussiaRico-Yuste, Alberto from Universidad Complutense de Madriddisabled, SpainAbad Diaz De Cerio, AnaLi, JingAbd El-Aziz, Tarek MohamedLiaimer, AntonAbdallah, Mohamed F.Liberton, MichelleAboal, MarinaLightfoot, Jorge D.Abou-Mansour, ElianeLimay-Rios, VictorAbraham, AnnLim\u00f3n, M. CarmenAbraham, Wolf-RainerLin, Cheng-WenAbrah\u00e3o Nencioni, Ana LeonorLin, GuitingAbrunhosa, Lu\u00edsLin, Hsin-TangAccoroni, StefanoLin, Shih-Hua\u00c1d\u00e1m, T\u00f6lgyesiLindahl, LasseAdamo, ShelleyLingwood, Clifford A.Adams, SolomonLins, LaurenceAdams, VickiLitaker, WayneAdamski, ZbigniewLiu, Biing HuiAd\u00e1nyi, N\u00f3raLiu, ChangqiAdewole, DeborahLiu, QianqianAggarwal, MeghaLivaniou, EvangeliaAgha, RamsyLlewellyn, Lyndon E.Agriopoulou, SofiaLluna, Amparo GameroAhel, IvanLo, Samuel Chun-lapAichinger, GeorgLobato-Marquez, DamianAjani, PenelopeLobner, Doug C.Ajigboye, Olubukola O.Locatelli, MarcelloAlassane-Kpembi, ImouranaLoi, MartinaAlbrecht, Eric A.Lopes Fuly, Andr\u00e9Alcaide, Francisco EspejoLopes, AdrianaAleu, JosefinaLopez Navas, Ana I.Alisaac, EliasL\u00f3pez-Sanmart\u00edn, MonserratAllen, Kerry ClintLou, JianlongAlmeida, MarisaLouzao Ojeda, M. 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C.Zatsepin, TimofeiLee, Hsin-ChenZeigler, DanielLee, Min-HoZeleny, ReinhardLee, Moo-SeungZervou, Sevasti-KiriakiLee, SangheeZewdie, Ephrem TakeleLee, Tse-MinZhang, KaiLeggieri, Marco CamardoZheng, Cai-MeiLelario, FilomenaZheng, JieLemichez, EmanuelZherdev, Anatoly V.Leo, Jack ChristopherZhou, Carol L. EcaleLeonardi, AdrijanaZielonka, \u0141ukaszLeonelli, FrancescaZieniuk, Bart\u0142omiejLepiarczyk, EwaZimba, PaulLepoutre, AlexandraZingali, Russolina BenedetaLetek, MichalZingli, RussolinaLevesque, Am\u00e9lieZsarnovszky, AttilaLewczuk, Bogdan\u017buk, MagdalenaLewin, MattZuliani, Juliana PavanLewis, RichardZupko, IstvanLi, DeyaoZupo, ValerioLi, DongyingThe editors also express their sincere gratitude to the following reviewers for their precious time and dedication, regardless of whether the papers were finally published:"}
+{"text": "Paraquat, (PQ), an herbicide that can induce Parkinsonian-like symptoms in rodents and humans. The consumption of phytochemical-rich plants can reduce the risk of chronic illnesses such as inflammation and neurodegenerative diseases. The present study aimed to investigate the protective effects of pomegranate seed extract (PSE) and juice (PJ) against PQ-induced neurotoxicity in mice.Mice were assigned into 4 groups; three groups received PQ  twice a week for 3 weeks. Two of the PQ-induced groups pretreated with either PSE or PJ. Detection of phytochemicals, total phenolics, and total flavonoids in PSE and PJ was performed. Tyrosine hydroxylase (TH) level was measured in the substantia nigra (SN) by Western blotting technique. Striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were detected using high-performance liquid chromatography (HPLC). The levels of adenosine triphosphate (ATP), malondialdehyde (MDA), and the activity of the antioxidant enzymes were estimated in the striatum by colorimetric analysis. Striatal pro-inflammatory and anti-inflammatory markers using enzyme-linked immunosorbent assay (ELISA) as well as DNA fragmentation degree by qualitative DNA fragmentation assay, were evaluated. Real-time polymerase chain reaction (qPCR) assay was performed for the detection of nuclear factor kappa B (NF-\u043aB) gene expression. Moreover, Western blotting analysis was used for the estimation of the cluster of differentiation 11b (CD11b), transforming growth factor \u03b2 (TGF-\u03b2), and glial cell-derived neurotrophic factor (GDNF) levels in the striatum.Pretreatment with PSE or PJ increased the levels of TH in the SN as well as DA and its metabolite in the striatum that were reduced by PQ injection. PSE and PJ preadministration improved the PQ-induced oxidative stress via a significant reduction of the MDA level and the augmentation of antioxidant enzyme activities. PSE and PJ also significantly downregulated the striatal NF-\u043aB gene expression, reduced the PQ-enhanced apoptosis, decreased the levels of; pro-inflammatory cytokines, CD11b, and TGF-\u03b2 coupled with a significant increase of; interleukin-10 (IL-10), GDNF, and ATP levels as compared with PQ-treated mice.The current study indicated that PSE and PJ consumption may exhibit protective effects against PQ-induced neurotoxicity in mice.The online version contains supplementary material available at 10.1186/s12906-021-03298-y. Parkinson\u2019s disease (PD) is a chronic neurodegenerative disease , affectiRecent studies reported the participation of the environment to PD pathogenesis  while a 2+. When PQ2+ is injected into mice for at least 3\u20134\u2009weeks, it induces a loss of dopaminergic neurons [2+ undergoes redox cycling in the presence of NADPH hydroxylase on the microglia and is reduced to the monovalent cation PQ+. PQ+ is a DA transporter substrate and is accumulated in the dopaminergic neurons where it starts a new redox cycle intracellularly. Subsequently, superoxide anions are generated and reactive oxygen species (ROS) are produced in the dopaminergic neurons leading to oxidative stress-related cytotoxicity and neurotoxicity [Paraquat  is the most common herbicide that is used in the agricultural sector worldwide for weed control , 11. PQ  neurons . In the toxicity . Consequtoxicity , 15.ATP exerts a vital role in the exchange of energy in various biological systems. Moreover, it is found in every metabolically active cell. Therefore, the measurement of ATP levels can be used as an indicator of the functional integrity of dopaminergic neuronal cells in the dorsal striatum .It has been reported that glial cell-derived neurotrophic factor (GDNF) exhibits a prominent neurorestoration and neuroprotection in multiple neurodegenerative diseases, including PD.  It was rDespite the pharmaceutical treatments of PD may reduce the symptoms, they exhibit severe adverse effects that preclude their long-term use  and it iPunica granatum L.) fruit, with various groups of phytochemicals, exhibits many therapeutic properties including antioxidant, anti-inflammatory, anti-proliferative, anti-cancer, antimicrobial, neuroprotective, and anti-apoptotic consequences [Naturally occurring phytochemicals have been extensively used nowadays for the protection of the body from oxidative damage ascribed with free radicals, neuroinflammation, and DNA fragmentation especially in the brain tissue . It has equences , 22. Notequences . Phenoliequences . Despiteequences .Up to our knowledge, the current study is the first-ever to assess the potential antioxidant, anti-inflammatory, and anti-apoptotic capabilities of pomegranate juice (PJ) and seed extract (PSE) against PQ-induced Parkinsonian mice model.PQ dichloride (\u2265 98%) was bought from Sigma . The rest of the chemicals were of the highest analytical grade.Punica granatum L.) fruits were reaped at Fayoum City, Egypt in the months of September\u2013October, 2019. The coordinates of the city are latitude 16\u00b0 53\u2032 12.59\u201c N and longitude: 42\u00b0 33\u2019 23.99\u201d E following the degree minutes second (DMS) system. The plant material was authenticated by comparing it with the well-known herbarium specimens found at the Herbarium of Flora Researches Centre, Agriculture museum campus (CAIM), Dokki, Giza, Egypt.Pomegranate  at 25\u2009\u00b0C for 24\u2009h. The mixture was then filtered by 0.45\u2009\u03bc pore size filters. The ethanol was completely evaporated using a rotary vacuum evaporator at 40\u2009\u00b0C and the seed extract was kept at \u2212\u200920\u2009\u00b0C until use.The GC-MS technique was carried out using a GC (Agilent Technologies 7890A) edged with a mass-selective detector  supplemented with an apolar Agilent HP-5\u2009ms (5%-phenyl methyl poly siloxane) capillary column (30\u2009m\u2009\u00d7\u20090.25\u2009mm i. d. and 0.25\u2009\u03bcm film thickness). Helium was the carrier gas with a linear velocity of 1\u2009ml/min. The temperatures of the injector and the detector were adjusted at 200\u2009\u00b0C and 250\u2009\u00b0C, correspondingly. Split injection mode and the MS operating factors were adjusted according to Fathy and Drees . The comTPC was measured in PSE and PJ by using the Folin-Ciocalteu method as described by Velioglu et al. . Gallic 3) colorimetric assay according to Zhishen et al. [TFC of PSE and PJ was detected by using aluminum chloride  guidelines for Ethical Conduct in the Care and Use of Laboratory Animals and approved by the National Organization of Drug Control and Research (NODCAR), Egypt.+\u00a02\u2009\u00b0C) and subjected to 12\u2009h light/dark cycle throughout the entire study. They were given water ad libitum and fed with the standard laboratory diet. Mice were allowed to accommodate the laboratory condition 1 week before the beginning of the experiment.Adult male albino mice weighing 20\u201325\u2009g (8\u2009weeks old) were brought from the National Cancer Institute (NCI), Egypt. Animals were kept in conventional cages (polycarbonate) under standard conditions of ventilation, temperature , malondialdehyde (MDA), antioxidant enzyme activities, inflammatory cytokines, the cluster of differentiation 11b (CD11b), GDNF, and transforming growth factor-\u03b2 (TGF-\u03b2). Part of the separated striatum was stored at \u2212\u200980\u2009\u00b0C until required for DA and 3,4-dihydroxyphenylacetic acid (DOPAC) estimation and conducting the laddered DNA fragmentation assay. Another part was kept in RNA lysis buffer for conducting a quantitative polymerase chain reaction assay.At the end of the study, all mice were euthanized by decapitation under pentobarbital  anesthesia . The braBoth DA and DOPAC levels were determined by high-performance liquid chromatography (HPLC) according to Chi et al.  with somATP content was estimated in the striatum as stated by the manufacturer\u2019s protocol using ATP Colorimetric/Fluorometric Assay Kit .MDA is an end product of lipid peroxidation (LPO). It was calculated by using  in the tissue homogenate by the formation of thiobarbituric acid reacting substances (TBARS) following the method of Esterbauer and Cheesem .CAT activity was detected using a CAT assay kit  according to the instructions from the producer.SOD activity was measured using the SOD Activity Assay Kit  as stated by the protocol\u2019s instructions.GPx activity was tested using the GPx Activity Colorimetric Assay Kit  according to the protocol\u2019s instructions. In this assay, GPx diminishes Cumene Hydroperoxide while oxidizing GSH to GSSG. Then it was reduced to GSH with NADPH depletion .Apoptotic DNA fragmentation was detected in the striatum according to the protocol described by Sambrook et al. . BrieflyThe levels of tumor necrosis factor (TNF)-\u03b1, interleukin (IL)-1\u03b2, IL-6, IL-10, were estimated in the tissue supernatant by sandwich enzyme-linked immunosorbent assay (ELISA) kits specific for mice  following the product\u2019s instruction. The absorbance was detected at 450\u2009nm using the microplate reader.RNA was isolated from the striatal tissue and qPCR was performed as stated by the instructions from the manufacturer using SV Total RNA Isolation System  and Applied Biosystems StepOne\u2122 , correspondingly according to Fathy and Said . The NanWestern blotting technique was performed to measure the protein level of TH in the SN. It was also conducted to estimate the protein expression levels of CD11b, TGF-\u03b2, and GDNF in the striatum. Equal amounts of total proteins (30\u2009\u03bcg) from the supernatant samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes . Membranes were blocked, incubated with 1:1000 of the specific primary antibody at 4\u2009\u00b0C overnight. Antibodies for TH , TGF-\u03b2 , GDNF , and CD11b  were used. Afterward, membranes were incubated with 1:5000 of goat anti-rabbit IgG antibody, HRP  or goat anti-mouse IgG, HRP  for 1\u2009h at room temperature. Detection reagents for enhanced chemiluminescence  were applied and the bands were detected using a chemiluminescence system .P\u2009<\u20090.05 among groups was considered statistically significant.Statistical analysis of data was performed using GraphPad PRISM . Data were expressed as the mean\u2009\u00b1\u2009standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey\u2019s test. Value of Table\u00a0Table\u00a0It was found that TPC in PSE is 655.00\u2009\u00b1\u200911.36\u2009mg of gallic acid equivalents/100\u2009g dry weight (DW) whereas TPC in PJ is 241.1\u2009\u00b1\u20099.99\u2009mg of gallic acid equivalents/100\u2009ml of juice.Regarding TFC, it was found to be 145.70\u2009\u00b1\u20096.65\u2009mg of catechin equivalents/100\u2009g DW of PSE whereas TFC in PJ is 39.40\u2009\u00b1\u20094.26\u2009mg of catechin equivalents/100\u2009ml of the juice.P\u2009<\u20090.001). Oral treatment of either PSE or PJ led a remarkable increase in the protein expression of TH (P\u2009<\u20090.001) when compared with the PQ -treated mice -induced group when compared with the control group P\u2009<\u20090.00. Oral trP\u2009<\u20090.0001). Pretreatment with PSE was associated with a significant elevation in DA and DOPAC levels (P\u2009<\u20090.001) when compared with PQ -induced mice. Administration of PJ also led to a conceivable increase in the levels of DA and DOPAC as compared to PQ -treated mice  in relation to PQ -induced mice.ATP level, the cell energy marker, significantly declined in the striatum after PQ injection when compared with the saline-injected group  except for PJ effect on CAT activity that exhibited a non-remarkable difference when compared with the PQ -treated group . A significant downregulation of NF-\u043aB gene expression was detected after oral administration of PSE or PJ, when compared with PQ -treated mice (P\u2009<\u20090.0001).Figure\u00a0P\u2009<\u20090.0001; Fig.\u00a0P\u2009<\u20090.0001; Fig. P\u2009<\u20090.0001; Fig. Figure\u00a0Punica granatum L.) contains variable phytochemicals that were referenced to exert antioxidant and anti-inflammatory impacts [Punica granatum L. against the PQ-induced PD model has not been explored yet.Different reports illustrated the relation between dietary supply and improved health problems. A close relation between vegetables and fruits\u2019 intake with the reduced risk of various health disorders such as; diabetes, strokes, cancers, and neurodegenerative diseases, was elucidated . Pomegra impacts . Althoug impacts , the proIn the present article, TH level substantially reduced in the SN following PQ treatment while this decline was markedly prevented by PSE or PJ pretreatment. The reduction in TH protein and DA levels by different mechanisms might induce the propagation of the movement disorders during the process of dopaminergic cell death . SubsequIn the current study, the levels of DA and its metabolite, DOPAC, markedly reduced following PQ treatment. Treatment with PQ was reported to reveal variable impacts on DA and DOPAC levels in different brain regions . HoweverATP is an important indicator of the energy exchange and the functional integrity for all active cells and its measurement can be used as a clue for detecting dopaminergic neuronal loss . It has Excessive accumulation of oxidatively damaged molecules or oxygen free radicals was prominent in various disorders, including neurodegenerative diseases such as PD.  In the pDrosophila melanogaster via the oxidative stress-induced pathway [Drosophila melanogaster. In the present experiment, PSE and PJ constituents preserved the neuronal structure in the striatum by prohibiting chromatin condensation. The abovementioned effect might be attributed to the counteracting antioxidant phytochemical components along with the total phenolic and flavonoids that were detected in both PSE and PJ.Apoptosis or programmed cell death was potentiated following PQ administration into  pathway . The mol pathway . Moreove pathway . In the  pathway  who showDifferent chronic diseases including neurodegenerative pathologies are correlated with the activation of chronic inflammation  with perActivation of the transcription factor; NF-\u043aB elicits an inflammatory response and implies neuronal integrity and signaling . In the Besides, NF-\u043aB activation has an essential pro-apoptotic role in PD and was correlated with the dopaminergic cell destruction in the 6-hydroxydopamine (6-OHDA) induced PD model of rats . ConsequThe current results demonstrated the levels of trophic factors; TGF-\u03b2 and GDNF in PQ-induced mice and their levels after treatment with either PSE or PJ. The level of TGF-\u03b2 significantly increased while GDNF substantially declined after PQ treatment. It has been reported that TGF-\u03b2 is expressed in the dopaminergic system pathway since the embryonic stage of development and its mRNA is upregulated as a result of the exposure to different lesions . ContradIt was recorded that GDNF is an important factor for dopaminergic neuronal survival  and thatPolyphenols include various groups that were classified based on the number of phenol rings and the chemically attached groups to them. The most crucial group of polyphenols is named as flavonoids that contain compounds such as vitexin, isovitexin, and flavones. The abovementioned components were recorded in the present work in PSE and PJ Tables\u00a0 & 2. OthIn conclusion, PQ injection supressed the TH level in the SN which confirms the hallmark feature of PD that was prevented upon pretreatment with either PSE or PJ. Furthermore, oxidative damage effect following PQ treatment might promote inflammatory reaction which induced DNA fragmentation with the decreased DA and DOPAC secretions along with the reduced ATP level in the striatum. Treatment with either PSE or PJ showed a protective impact against PQ-induced toxicity in the striatum through protection from the oxidative stress, inhibition of inflammation, rescuing the cells from DNA fragmentation, and restoration of ATP level. Thus PSE and PJ supplementation may be useful therapeutic agents that protect from PD.Additional file 1.Additional file 2.Additional file 3."}
+{"text": "Bacterial and fungal infections, such as skin and soft tissue infections (SSTIs) and infective endocarditis (IE), are increasing among people who use drugs in the United States. Traditional healthcare settings can be inaccessible and unwelcoming to people who use drugs, leading to delays in getting necessary care. The objective of this study was to examine SSTI treatment experiences among people utilizing services from syringe services programs. This study was initiated by people with lived experience of drug use to improve quality of care.We conducted a cross-sectional survey among participants of five syringe services programs in North Carolina from July through September 2020. Surveys collected information on each participant\u2019s history of SSTIs and IE, drug use and healthcare access characteristics, and SSTI treatment experiences. We examined participant characteristics using counts and percentages. We also examined associations between participant characteristics and SSTI history using binomial linear regression models.Overall, 46% of participants reported an SSTI in the previous 12\u00a0months and 10% reported having IE in the previous 12\u00a0months. Those with a doctor they trusted with drug use-related concerns had 27 fewer  SSTIs per every\u00a0100 participants\u00a0compared to those without a trusted doctor. Most participants with a SSTI history reported delaying (98%) or not seeking treatment (72%) for their infections. Concerns surrounding judgment or mistreatment by medical staff and self-treating the infection were common reasons for delaying or not seeking care. 13% of participants used antibiotics obtained from sources other than a medical provider to treat their most recent SSTI. Many participants suggested increased access to free antibiotics and on-site clinical care based at syringe service programs to improve treatment for SSTIs.Many participants had delayed or not received care for SSTIs due to poor healthcare experiences. However, having a trusted doctor was associated with fewer people with SSTIs. Improved access to non-judgmental healthcare for people who use drugs with SSTIs is needed. Expansion of syringe services program-based SSTI prevention and treatment programs is likely a necessary approach to improve outcomes among those with SSTI and IE. Staphylococcus aureus [Bacterial and fungal infections are increasing among people who use drugs (PWUD) in the United States \u20133. Theses aureus \u201314 and hs aureus \u20137, 15.PWUD face multiple barriers when accessing traditional medical services resulting in a long-standing history of mistrusting formalized medical care . PWUD ofThe objective of this study was to describe and quantify personal and healthcare experiences with, as well as treatment strategies for, SSTIs and IE among PWUD utilizing SSP services. It also aimed to understand the reasons PWUD have for alternative treatment strategies and how SSPs might better meet the needs of PWUD with SSTIs.This cross-sectional health needs assessment was initiated by the SSPs in response to increasing concerns about SSTIs among the populations served by SSPs. To recruit survey sites, staff from one SSP (North Carolina Survivors Union) contacted other SSPs using a statewide contact list. Among approximately 30 SSPs in North Carolina at the time of the survey, four additional programs agreed to participate, resulting in a total of five survey sites.Surveys were administered from July through September 2020. During this time, the coronavirus pandemic (COVID-19) precautions resulted in reduced SSP operations for some sites and shorter durations of engagements with SSP participants, although sites reported that demands for services had increased since pandemic onset. We used a convenience sampling approach to identify survey participants. Specifically, SSP staff approached persons seeking services  and asked about their interest in participating in a short survey. Surveys were self-administered at the SSP via electronic tablets using a standardized instrument . The medhttps://doi.org/10.17615/4g3h-j073. Demographic characteristics included age, gender, race, and ethnicity. Healthcare access measures included health insurance coverage and access to a trusted doctor for drug use-related concerns. SSTI and IE history was defined by those events occurring ever in the participant\u2019s lifetime or within the previous 12\u00a0months. To improve the accuracy of questions relating to SSTI history, the survey tool asked participants whether they would be willing to see photos of several types of SSTIs to aid recall. Based on this response, participants were either shown these photos in the survey tool prior to the SSTI history questions or automatically proceeded to these questions without displaying the photos.All survey measures were self-reported by survey participants. The full survey is available at Participants were asked about injection of specific drugs, including cocaine, crack-cocaine, fentanyl, heroin, methamphetamine, and prescription opioids, in their lifetime and during the 12\u00a0months prior to survey administration. Participants were also asked about whether they had missed a shot  or skin popped  in the previous 12\u00a0months. Participants were asked about SSTI treatment experiences including treatment strategies  and barriers to treatment . Participants were also asked to provide suggestions to improve their\u00a0SSTI treatment experiences.We examined frequencies and percentages of the study population\u2019s overall drug use and healthcare access characteristics by SSTI and IE history. We used unadjusted and adjusted binomial linear regression models to estimate prevalence differences and corresponding 95% confidence intervals for potential predictors  of SSTI history within the previous 12\u00a0months. Participants with missing values for any of the specific variables included in each model were excluded . Prevalence differences can be interpreted as the excess or reduced number of people with SSTIs per\u00a0every\u00a0100 people\u00a0when comparing one group to another . Adjusted models included indicator variables for self-reported race , gender , and health insurance coverage (dichotomized as yes or no). Adjusted models evaluating health insurance as a potential predictor only included indicator variables for race and gender. All statistical analyses were conducted using SAS 9.4 .Among those approached at the SSPs, individuals who\u00a0displayed an interest in completing the survey were provided with a brief description of the survey purpose and intended use of the data. After reviewing this information, these\u00a0individuals were asked if they would still\u00a0be willing to participate. No compensation was provided. This study was reviewed and approved by the University of North Carolina at Chapel Hill Institutional Review Board. People with lived experience of drug use were involved in survey conceptualization and design, questionnaire development, data collection, and interpretation of results.Of the 281 people approached, 37% (n\u2009=\u2009105) agreed to participate in the survey. Response proportion varied from 17 to 71% by site, with each site contributing between 11 and 36 participants. Of the 105 participants, 51% (n\u2009=\u200952) were men and 87% (n\u2009=\u200989) were white. The median age of participants was 39\u00a0years-old . A total of 34% (n\u2009=\u200935) of participants had health insurance coverage and 29% (n\u2009=\u200930) had a doctor they trusted with health concerns relating to drug use  of participants had a lifetime history of SSTIs and 46% (n\u2009=\u200947) had a history of SSTI in the previous 12\u00a0months. Among those with a lifetime history of SSTIs, most reported one  or multiple SSTIs  in the previous 12\u00a0months. Seventeen percent (n\u2009=\u200917) of participants had been told by a doctor that they had IE in their lifetime and 10% (n\u2009=\u200910) were told this in the previous 12\u00a0months. Of the 17 respondents with a lifetime history of IE, eight (47%) had surgery for the infection and five (29%) reported they were living with untreated IE. The proportion of participants with a history of SSTI in the previous 12\u00a0months varied by survey site, from as low as 35% (n\u2009=\u200912) at one site to as high as 92% (n\u2009=\u200911) at another site.Adjusted models accounted for gender, race, and insurance status. Cocaine injection was associated with an increase in recent SSTI\u00a0relative\u00a0to other substances injected: There were 17 more people with SSTI\u00a0per every\u00a0100 participants\u00a0 who injected cocaine\u00a0compared to\u00a0those who had not injected cocaine in the previous 12\u00a0months  with access to a doctor they trusted compared to those without\u00a0access to a doctor, after adjusting for gender, race, and health insurance status.Among the 65 participants with a lifetime history of SSTIs, 71% (n\u2009=\u200946) reported having a SSTI that did not resolve on its own. In order to treat a SSTI, 63% (n\u2009=\u200939) reported taking antibiotic pills that were not prescribed to them.Of those experiencing a SSTI that did not resolve on its own, nearly everyone reported delaying  or not receiving treatment 72%, n\u2009=\u200933) for their SSTI. Participants cited judgment or mistreatment from medical staff , self-treatment , time commitments , legal concerns , and previous bad experiences\u00a0when getting treatment  as main reasons for delaying or not receiving treatment  had sought help from doctors or friends within a few days to a few weeks after first noting the infection. The most common sources of treatment included healthcare providers , and friends or family members  . Delaying or not receiving treatment was also related to practical concerns, such as time constraints, financial cost, and transportation, all of which impacted accessibility to clinical care. In addition, many participants sought treatment from sources outside of traditional healthcare settings, such as the use of antibiotics obtained from sources other than healthcare providers and treatment from friends and family.The burden and severity of SSTIs and IE underscores the need for treatment and prevention innovation. These two infections are closely related. 60% of our participants with a history of IE in the previous 12\u00a0months also had a history of SSTIs in the previous 12\u00a0months. Prevention of SSTIs, such as harm reduction education\u00a0and access to safe drug use supplies, may also lead to an individual\u2019s subsequent reduced risk of IE. Stigma and mistreatment by medical staff are common barriers to care for PWUD \u201320. Resp64% of participants in our survey had a lifetime history of SSTIs. Comparable studies have found similarly high prevalence estimates , 6, 8. PIn line with our results,\u00a0other research has also identified that some people obtain antibiotics from sources other than healthcare providers to treat SSTIs; however, recent research has been qualitative and recent estimates of this type of antibiotic use are not established , 23. ForThis study has several notable strengths. Our study team included people with lived experience of drug use who were involved in conceptualization and design, survey development, and results interpretation. Specifically, the initiation of this needs assessment by people with lived experience of drug use led to a timely and impactful topic with relevant and culturally appropriate survey questions. Our study team\u2019s structure provided a platform to quickly contextualize results and translate how the results can inform practice and advocacy efforts. This study also included five SSPs from different regions of North Carolina, representing a wide\u00a0range of PWUD populations and SSPs. In addition, much of the existing research surrounding SSTI treatment among PWUD has focused on self-treatment strategies. Our study adds to this previous research by identifying specific barriers to clinical treatment and incorporating PWUD recommendations to improve SSTI treatment.Some limitations should be noted. First, these results are not intended to be generalizable to all PWUD seeking services; selection bias arising from non-participation and convenience sampling could not be avoided while complying with COVID-19 social distancing mandates. In addition, survey participation varied by SSP which may be due to the type of site . While stigma and privacy concerns could be drivers of non-participation, we believe this was minimized due to SSP staff administering the surveys as opposed to outside researchers. In addition, we were unable to provide incentives for study participation. Future studies should consider providing incentives to be respectful of participants\u2019 time and increase participation. Second, we did not collect information on the participant injection frequency, which is likely a major predictor of SSTIs and other injection-related injury and infections . Third, Improved access to quality, non-judgmental healthcare is needed for PWUD with SSTIs. Future research should examine which specific treatment approaches are\u00a0beneficial, preferred, and well-accepted by PWUD. Additionally, education about SSTI prevention should be, and often is, routinely offered at SSPs. Similar harm reduction education should be offered in healthcare settings that serve PWUD with SSTIs or PWUD at risk of developing SSTIs. Additional resource\u00a0allocation, such as funding for wound care programs and SSP-based clinical services, is likely an\u00a0important strategy to prevent these infections and improve outcomes among PWUD with SSTIs and IE."}
+{"text": "Background. The peak of sexually transmitted infections (STI) among adolescents/young adults suggests a low level of prevention. In order to assess whether the level of sexual health education (SHE), received by several channels, was effective at improving sexual behaviors, we conducted a survey among freshmen from four Italian universities. Methods. This observational cross-sectional study was carried out with an anonymous self-reported paper questionnaire, administered during teaching lectures to university freshmen of the northern  and southern  parts of the country. Knowledge of STI , knowledge of STI prevention (dichotomous variable: yes vs. no) and previous STI occurrence  were the outcomes in the statistical analysis. Results. The final number of freshmen surveyed was 4552 (97.9% response rate). The mean age of respondents was 21.4 \u00b1 2.2 years and most of them (70.3%) were females. A total of 60% of students were in a stable romantic relationship. Only 28% respondents knew the most effective methods to prevent STI , with a slightly higher prevalence of correct answers among females (31.3%) than males (25.8%). Students with history of STIs were 5.1%; they reported referring mostly to their general practitioner (GP) (38.1%) rather than discussing the problem with their partner (13.1%). At multivariable analysis, a significantly higher level of STI knowledge was observed in older students (25+ years of age), biomedical students, and those from a non-nuclear family; lower levels were found among students of the University of Palermo, and those who completed a vocational secondary school education. Those who had less knowledge about the most effective tools to prevent STIs included males, students from the University of Palermo, students registered with educational sciences, economics/political sciences, those of foreign nationality, and those whose fathers had lower educational levels. The risk of contracting a STI was significantly lower only in students not in a stable relationship , whereas such risk was significantly higher in students with higher STI knowledge . Discussion and Conclusions. University freshmen investigated in this study had poor knowledge of STIs and their prevention. Unexpectedly, those with higher levels of knowledge had an increased risk of STIs. There have been no educational interventions\u2014with good quality and long-term follow-ups\u2014that increased the confidence that such SHE programs could have population level effects. A new high-quality study is therefore recommended to assess the effectiveness of an intervention generating behavioral changes; increasing only STI knowledge may not be sufficient. Sexually transmitted infections (STIs) are considerable public health threats across all ages in high-income countries, particularly among teenagers . AccordiMore than 30 different bacteria, viruses, and parasites are known to be transmitted via vaginal, anal, and oral sex, while some can be spread through blood or blood products. Many STIs, such as syphilis, HIV, hepatitis B, chlamydia, gonorrhea, and herpes, can also be transmitted from mother-to-child during pregnancy and childbirth . STIs caDespite making up approximately 25% of the sexually active population, individuals aged 15\u201324 account for half of the 20 million incident cases of STIs reported annually in the USA .Modifying risk-taking behaviors is a difficult task, especially in younger ages . AccordiSexual health education (SHE) is grounded in internationally recognized human rights and it plays a central role in the preparation of young people to achieve safe, productive, and fulfilling lives . SHE is In March 2017, political pressures prompted the Department of Education of the British government to announce that sex and relationship education (SRE) would have to be officially introduced in British primary schools by September 2019 . SRE enaNotwithstanding this progress, three recent literature reviews reported evidence of a different understanding on the issue.A comprehensive review of school-based interventions to improve the sexual-health of adolescents was published in 2017. Authors have systematically identified and synthesized evidence-based recommendations across 37 reviews, from 224 relevant trials, and quasi-experimental studies, including the highest quality and the most highly cited reviews in the field of sexual health. Methodological weaknesses\u2014including lack of randomization, insufficient follow-up, small sample sizes, and lack of replication studies\u2014were identified by many reviewers, along with weak and inconsistent evidence on behavioral effects. However, a clear conclusion could also be drawn. Sexual-health and relationship interventions focusing exclusively on sexual abstinence were not effective in modifying the sexual behaviors of school students .p = 0.22; n = 6 studies; 12,633 participants; I2 = 77%; low-quality evidence) as well as cannabis use or unhealthy diets [An intervention review on randomized controlled trials (RCTs), including cluster RCTs, examined the effectiveness of universal school-based interventions targeting multiple risk behaviors among children and young people up to 18 years of age. Among a total of 34,680 titles identified, 27,691 articles screened, and 424 full-text articles assessed for eligibility, a total of 70 eligible studies were included in the review. The evidence was strongest for the efficacy of universal school-based interventions in preventing tobacco use, alcohol misuse, consumption of illicit drugs, antisocial behavior, and in improving physical activity among young people. In contrast, the evidence was less conclusive for the effect of health promotion interventions on risky sexual behaviors  .Despite most sexual health studies targeting secondary school students, first year university students represent an important group in this context, because they are young adults, attempting to be independent from their families, usually already sexually active, and keen to new sexual experiences. Although SHE programs are no mandatory in Italian schools, students can receive SHE in many other ways. This can include, for example, direct experience, peer group, friends and sexual partners, external courses or activities, university courses, or online resources ,20.The above-mentioned literature gap requires further evidence. Therefore, the main objective of this study was to explore the sexual health knowledge (SHK) of first year students from four Italian universities, investigating whether their knowledge, reflecting SHE received by different channels, was effective at preventing STIs.We ran a cross-sectional observational study on freshmen at four Italian universities from the northern  and southern  part of the country. The final number of freshmen surveyed was 4552 (97.9% response rate). Subjects were recruited by convenience sampling based on individual negotiations with the respective academic staff. Students enrolled in educational sciences, foreign language/literature, biology, chemistry, medicine, natural sciences, engineering, architecture, political sciences and economics were selected to participate in this survey.The survey was conducted in 2012 via an anonymous self-reported paper questionnaire. Since there was no validated tool, a new questionnaire  was given to the students to complete the questionnaire.The questionnaire was not intended as a psychometric instrument with Likert-scale or Likert-type items; therefore, Cronbach tests were not performed. The tool collected plain descriptive information, such as personal data , family data , and information on their current romantic relationships . Furthermore, several multiple-choice questions were recoded as follows.Please indicate which of the following diseases can be transmitted by sex\u201d. The multiple answer options were the following: \u201cGonorrhea; Mononucleosis; Syphilis; AIDS; HPV (Human Papilloma virus); Hepatitis C (HCV); Hepatitis B (HBV); Hepatitis A (HAV); Genital Herpes; Streptococcal infection; Venereal lymphogranuloma; Wart; Psoriasis; Candidiasis; Tuberculosis\u201d. The interviewees had to answer whether each of the above diseases were:Sexually transmittable;Non sexually transmittable; orWhether (s)he did not know.Question 78 asked, \u201cSexually transmittable\u201d. In case the student answered, \u201cNon sexually transmittable\u201d or \u201cI do not know\u201d, a value of 0 was assigned to the respective disease. All values were then summed up to create a linear numerical score ranging from 0 to 15, which was used as outcome 1 in the statistical analysis.With the exception of psoriasis (negative control), all of the above were STIs. A value of 1 was assigned each time the above diseases were considered, \u201cWhich of the following are the most efficacious methods to prevent sexually transmitted infections?\u201d The multiple answer options were the following: basal temperature thermometer; oil; anti-contraceptive pill; billing\u2019s method; morning after pill; diaphragm; interrupted coitus; spermicide; condom; transparent membrane; abstinence; contraceptive plaster; male pill; ulipristal acetate; cervical cap; vaginal washing; contraceptive sponge. Interviewees could provide up to four answers, which could either be:Correct answers, where, \u201csexual abstinence\u201d and \u201ccondom\u201d (simultaneously) were indicated, even if additional options were ticked.Wrong answer: any other combination.Question 76 asked, \u201cKnowledge of STI prevention became a dichotomous variable coding 0 (wrong answer) or 1 (correct answer), which was used as outcome 2 in the statistical analysis.What did you do when you contracted a STI in the past?\u201d Up to three different answers could be chosen on the questionnaire, among the following:I asked a friend for advice;I referred to a consultant gynecologist;I tried to solve it on my own;I went to my general practitioner (GP);I went to accident and emergency (A&E);I discussed the issue with a family member;I discussed the issue with my partner.Students were also asked (question 69) whether they had ever contracted a STI. There were three pre-classified answers: \u201cNo\u201d (coded as 0); \u201cDon\u2019t know\u201d (code = 1); and \u201cYes\u201d (code = 2). Those who answered \u201cYes\u201d were then asked, \u201cAnswers to question 69, on whether students had ever contracted a STI, were coded as a three-level polytomous variable that was used as outcome 3 in the statistical analysis.No\u201d was used as the baseline comparison group (a \u201cpivot\u201d) and the other categories were separately regressed against the pivot outcome. The regression results were displayed in terms of relative risk ratios (RRR) with the corresponding 95%CI.Outcome 1 (STI knowledge) was employed as a dependent variable in a multiple linear regression model (model 1). Outcome 2 (STI prevention knowledge) was the dependent variable in a multiple logistic regression model (model 2). The predictors were all factors  shown in p < 0.05 was the criterion\u2014in order to reduce the confounding bias. Missing values were excluded and a complete case analysis was performed.In all models, backward stepwise selection of variables was used\u2014In order to increase their representativeness, results of the multivariable regression analyses were weighted for the distribution by age and sex of the Italian census population of 2011.STATA 14.2  was used as the statistical software package.Approval to conduct the study was obtained from the Research Ethics Committee (REC) of the University of Padua (Prot. REC-UNIPD 7/5/2020) and the research methodology was in accordance with the relevant guidelines and regulations. Since all students were 18 years of age or older, parental consent was not required. Informed consent for study participation was obtained from the study subjects.The final number of university freshmen recruited by the survey was 4552 (97.9% response rate).A total of 83.7% of the respondents had a nuclear family; 18.6% reported being a single child, 53.4% had one sibling, 22.0% two siblings, and 6.1% three or more siblings. A total of 3.3% of students had a mother of foreign nationality, while 2.7% had a father of foreign nationality. Moreover, 18.0% of the students\u2019 fathers and 16.0% of students\u2019 mothers had a postgraduate education. A total of 34.4% of fathers had educational levels limited to lower secondary schools, while the respective proportion for mothers was 34.6%.A total of 59.5% of interviewees received classical/scientific secondary school education or already had a university degree, whereas 13.7% had a secondary education in art, foreign languages, or socio-pedagogical classes, and 26.8% came from a vocational secondary school. The distribution by university was as follows: University of Padua 40.5%; University of Palermo 38.3%; University of Bergamo 14.7%; and the University of Milan 6.6%). Among university campuses, the distribution of students by undergraduate course of study was fairly homogeneous. However, no students of the course of literature/foreign language were recruited from the University of Padua. By contrast, students from the University of Bergamo were mainly enrolled in literature/foreign language courses (data not shown).There were 60% of students in stable relationships\u201465.8% (=2088/3174) among females and 46.4% (=618/1333) among males. The percentage of stable relationships increased with the age of the interviewees: 56.9% among students 18\u201320 years old; 61.6% among those 21\u201324 years old; 68.9% in students 25 or older (data not shown). Among those in a romantic relationship, 90% described themselves as being in love with their respective partners, 9% did not know, and only 2% described themselves as not being in love with their partners. Stable relationships were ongoing for at least 24 months in 52% of cases, 13\u201324 months in 21% of cases, 7\u201312 months in 12% of cases, and 3\u20136 months in 81% of cases.Which of the following diseases can be transmitted by sex?\u201d To identify areas that could benefit increasing SHE, STIs were arranged in I do not know\u201d (DK). More than half of the students  did not know that streptococcus, lymphogranuloma venereum, and hepatitis A belong to STIs. Among males, a percentage >50% did not know that candida and HPV are STIs. On the other hand, 97.2% of respondents recognized AIDS as a STI. A total of 84% of students were aware of the sexual transmissibility of herpes genitalis, with rather consistent distributions by sex and the university campus. A total of 80% of respondents recognized syphilis as a STI, although with a lower proportion (70%) among students from the University of Palermo. Knowledge of HBV and HCV as STIs displayed a distribution fairly consistent across different universities. Gonorrhea was indicated as a STI by 45% of students and lymphogranuloma venereum (chlamydia) by 32.1%, with alike distribution by university and sex.Which of the following are the most efficacious methods to prevent sexually transmitted infections?\u201d It can be noted that, as many as 86% of the respondents  knew about the use of condoms in preventing STI. Sexual abstinence was reported as the second most effective option to prevent STIs after condoms, with a fairly consistent distribution across different university campuses and by sex of respondents.As can be seen from Undergraduate course of study\u201d. Always using the group of biology/medicine/chemistry/natural sciences as reference categories, all other undergraduate courses showed significantly lower knowledge on outcomes 1 and 2; in particular, freshmen in educational sciences showing the lowest knowledge of STI  and STI prevention . The second example was \u201cuniversity campus\u201d. Using as reference the University of Padua, freshmen at the University of Palermo were found to have significantly less knowledge of both STI  and STI prevention , but, again, they were not at risk of STI.Few variables had an effect on both outcomes. An example was \u201cThe remaining variables had one-to-one effects. Knowledge of STI was higher in students aged 25+ years  compared with those aged <21 years, and in those from a non-nuclear type of family ; whereas it was lower among students who completed a vocational secondary school education . Knowledge on the most efficacious preventative tools against STIs was significantly lower in males , in students of foreign nationality , in those whose fathers had their education limited to secondary school  or junior secondary school , or who were employed as technicians .Yes\u201d) or not fully aware (first column heading = \u201cDon\u2019t know\u201d) of having contracted a STI in the past were compared with the same base \u201cNo\u201d . A different epidemiologic pattern was found. In the first column of Yes\u201d, a value of RRR < 1  was observed in male students , in those whose fathers were under 50 years of age , and among interviewees not in a relationship , while the risk of contracting a STI increased with higher level of outcome 1 . The latter variable was added as a further predictor in model 3 of the multinomial regression. Knowledge of STI prevention (outcome 2) was also introduced as a predictor in model 3, but the variable was removed by the backward stepwise selection.The overall knowledge of STIs among sample students was low ;Students with histories of STIs referred mostly to their GPs (38.1%), followed by a consultant gynecologist (13.1%), a family member (15.1%), or an experienced friend (12.5%), whereas discussing the problem with their partner was reported only by 13.1% freshmen ;Only 28% of the students knew the most effective prophylactic device to prevent a STI ; the percentage being slightly higher for females (31.3%) than males (25.8%) ;Significantly lower levels of STI knowledge were found among males, students at the University of Palermo, those who completed a vocational secondary school education, and non-Italian nationals ;Categories of individuals significantly less knowledgeable on the most effective options in preventing STI included males, students at the university of Palermo, students in educational sciences, students in economics/political sciences, those of foreign nationality, those with two siblings, and those whose fathers had lower educational levels ;The risk of contracting a STI was found to be lower in males, in students with fathers aged < 50 years and in students not in a stable relationship; by contrast it increased with higher knowledge of STIs .The present survey evidenced that:While HIV and syphilis were correctly recognized as STIs by 97% and 80% respondents in the present survey, gonorrhea was correctly identified by 44.9% of them and Chlamydia (lymphogranuloma venereum) by 32.1%, with rather similar distribution by university campus and sex of respondents. Genital warts, correctly recognized as STI by 15.0% of interviewees, was by far the most unknown STIs in our survey.A systematic review on young European adolescents aged 13\u201320 years reported a relatively higher knowledge of HIV/AIDS that has been probably influenced by the extensive preventative and informative campaigns deployed at international level over the last 30 years ,22,23,24I don\u2019t know\u201d (DKs) were added to negative answers, TB becomes the least disease associated with sex in the present survey. TB is not spread through sexual intercourse or kissing or other touch. TB bacteria are spread through the air when a person with TB coughs, sneezes, speaks, or sings. Several associations between the risk for TB and lifestyle factors have been identified. For example, unmarried persons are at higher risk of TB than married individuals. Moreover, HIV co-infection is the strongest known risk factor for disease progression from latent to active TB [If \u201cctive TB .The present study evidenced relevant cross-regional differences within Italy on SHK in terms of STIs prevention. In particular, students of the university of Palermo consistently showed significant lower levels of SHK, somehow confirming the cultural barriers for the implementation of SHE in Southern Italy , as alreAlthough condom was considered by far (86.2%) the most effective option against STIs in the present survey, with similar distribution by university campus and sex of respondents, only 28.0% of the students acknowledged both condoms as well as sexual abstinence as the most efficacious prophylactic tools to prevent STIs. Likewise, only 22% of 2867 students from the cities of Lecce and Genoa knew that both condoms and sexual abstinence were the most reliable methods to prevent STIs .partly safe\u201d versus \u201cvery safe\u201d to prevent STIs [In the study here presented, the contraceptive pill was the third most considered preventive option rated effective to prevent STIs; this was also confirmed by a survey amongst undergraduate biomedical students of Palermo . In the ent STIs . Likewisent STIs .In this study, only 13.1% of students who contracted a STI discussed the problem with their partners . HoweverIn the present study, biomedical students had a significantly higher knowledge and understanding of both STIs and the most effective ways to prevent them, a finding consistent with the existing literature ,35,36,37The cross-sectional design, without a time dimension in the present study, could not explain the paradoxical result of the increasing risk of STIs with higher STI knowledge. Although it is well known that gaps exist between knowledge of STIs and good preventive sexual practices, it seems more likely that SHK was primarily gained by direct experience of STIs, rather than from acquiring knowledge to prevent STI infection.Only 13% of students with history of STIs reported discussing the problem with their partners, thereby increasing the risk of spreading the infection. The lower risk of past STIs among males indicated that, in the present study, STIs were transmitted mainly by heterosexual contact or lesbian sex. The key to preventing the spread of STIs, especially in epidemics sustained mainly by heterosexual transmission, is modifying sexual behaviors. Multiple  sexual partnerships, combined with unprotected sex, is a critical risk factor for the spread of STIs .About 40% of our students were not in a stable relationship, and sexual abstinence is a key factor to contain the risk of STIs, since young people aged 16\u201324 years are more likely to report a new or two or even more sexual partners of the opposite sex in the past year .be faithful\u201d implies monogamy, it also includes reductions in casual and multiple sexual partnerships. Behavior changes to prevent STIs have mainly promoted condom use or abstinence, whereas the containment of the number of sexual partners remains a neglected component of ABC [The so called \u2018ABC\u2019 approach, where \u201cA\u201d stands for abstinence or delay of sexual activity, \u201cB\u201d for be faithful to one partner, and \u201cC\u201d for condom use, is receiving growing interest . Althougt of ABC . Albeit,t of ABC ,40. Liket of ABC , intervet of ABC .Recent surveys reported that the first sexual intercourse experiences occurred at the average age of 15.6\u201316.0 years among Italian adolescents, frequently without protection . For thiservices or clinics provided in schools; services located near schools that conduct outreach work within those schools; or services located near schools that liaise formally with those schools. The interventions of interest are those delivered to individuals who attend the services on a voluntary basis, and do not include either classroom interventions\u201d [should have access to free, confidential, adolescent-responsive, and non-discriminatory sexual and reproductive health services, information and education\u201d [School-based/linked sexual-health services are \u201centions\u201d . Accordiucation\u201d . Most EUucation\u201d ,43. For ucation\u201d .The creation of sexual health clinics implies instituting specialist training programs for sexual health professionals who should cooperate with families, school teachers, local pharmacies and other allied health professionals  to promote sexual health within an integrated multi-agency framework .The level of SHK among first year students at four Italian universities was ineffective at preventing STIs. Therefore, a new high-quality study is warranted to assess the effectiveness of preventive sexual health interventions, promoting behavioral changes among adolescents and young adults, since only improving their SHK may not be sufficient."}
+{"text": "Divergence time estimation is fundamental to understanding many aspects of the evolution of organisms, such as character evolution, diversification, and biogeography. With the development of sequence technology, improved analytical methods, and knowledge of fossils for calibration, it is possible to obtain robust molecular dating results. However, while phylogenomic datasets show great promise in phylogenetic estimation, the best ways to leverage the large amounts of data for divergence time estimation has not been well explored. A potential solution is to focus on a subset of data for divergence time estimation, which can significantly reduce the computational burdens and avoid problems with data heterogeneity that may bias results.In this study, we obtained thousands of ultraconserved elements (UCEs) from 130 extant galliform taxa, including representatives of all genera, to determine the divergence times throughout galliform history. We tested the effects of different \u201cgene shopping\u201d schemes on divergence time estimation using a carefully, and previously validated, set of fossils. Our results found commonly used clock-like schemes may not be suitable for UCE dating (or other data types) where some loci have little information. We suggest use of partitioning  and selection of tree-like partitions may be good strategies to select a subset of data for divergence time estimation from UCEs. Our galliform time tree is largely consistent with other molecular clock studies of mitochondrial and nuclear loci. With our increased taxon sampling, a well-resolved topology, carefully vetted fossil calibrations, and suitable molecular dating methods, we obtained a high quality galliform time tree.We provide a robust galliform backbone time tree that can be combined with more fossil records to further facilitate our understanding of the evolution of Galliformes and can be used as a resource for comparative and biogeographic studies in this group.The online version contains supplementary material available at 10.1186/s12862-021-01935-1. Divergence time estimation is fundamental to understanding many aspects of the evolution of organisms, such as character evolution, diversification, and biogeography , 2. Due Robust molecular dating depends on several factors, with the most critical being the use of suitable fossil calibration points, a broadly sampled and well-resolved phylogenetic topology, and adequate application of molecular clock methodologies . AlthougWell-supported phylogenies, based on loci throughout the genome, are now available for many groups. Whole and reduced representation genome sequencing methods  provide hundreds to thousands of loci throughout the genome and have significantly improved phylogenetic estimation , 13. ParHowever, while UCEs and other phylogenomic datasets show great promise in phylogenetic estimation, the best ways to leverage the large amounts of data for divergence time estimation has not been well explored. These datasets are large, which can place computational burdens on the programs used to estimate divergence time . More iThe Galliformes (gamebirds or landfowl) includes some of the best-studied avian species . Recent taxonomies , and molIn this study, we had three main goals. First, we wanted to estimate a robust phylogeny sampled broadly through the entire order by obtaining UCEs from at least one representative of each genus. Second, using this phylogeny, we wanted to test the effects of different \u201cgene shopping\u201d schemes on divergence time estimation using a carefully, and previously validated, set of fossils , 11, 31.We obtained UCEs from four outgroups and 130 galliform species , the second was the node between Alectoris and Perdicula\u2013Pternistis (62%), and the third one was within Pternistis (46%). The species tree, which was estimated using SVDquartets [Penelopina sister to all other guans  whereas the species tree united Penelopina with Chamaepetes  in the species tree  to reduce computation time. The ML tree estimated from the 75% complete matrix of the reduced set of 48 taxa was well-supported with only one node receiving less than 100% bootstrap support (97% bootstrap support for the node of p\u2009<\u20090.05) to other schemes when comparing all the 49 time points. Restriction to the most clock-like loci or most clock-loci partition sometimes yielded more recent divergence times, particularly for the deeper divergences  the 100% complete matrix (hereafter 100%); 2) the 95% complete matrix (hereafter 95%); 3) the most clocklike loci of the 95% complete matrix (hereafter 95%-loci-clocklike); (4) the most treelike loci (hereafter 95%-loci-treelike); (5) the most clocklike partition from PartitionFinder analysis  of the 9 the 95%  the mostp\u2009<\u20090.05).We found the alpha parameter and the average percent of informative sites were extremely low in the two clock-like and the largest partition schemes  formed a separate clade distinct from the burrow-building genera   Fig.\u00a0. While s support , 43), oubuilders . Howevere genera  rather tied Fig.\u00a0.Aburria, Chamaepetes Penelope, Penelopina, and Pipile). A recent study analyzed relationships among genera within Cracidae using UCEs (using the same data as in this study), mitochondrial sequences and nuclear introns [As noted in the results, relationships within Cracidae conflicted between the ML and species trees within the typical guan lineage  all had very low percentages of parsimony informative sites Table . CompareAnother common concern for phylogenetic analyses and, more specifically, molecular dating is variation among different loci . AlthougStruthio and Apteryx in our study) and improper prior distributions [The split between Anseriformes and Galliformes estimated from the 95%-PF-treelike scheme on the 135 taxa dataset is about 82.1\u00a0Ma, the CI of this node generally overlapped with recent analyses , 41, 42,ibutions , which aThe divergence times among the five galliform families were very similar to the ones estimated from mitochondrial and nuclear loci . HoweverIn Megapodiidae, the divergence time estimates from our study were older than those from mitochondrial and nuclear loci . Only onca. 13.1\u00a0Ma in that study vs. ca. 13.3\u00a0Ma in this study, which demonstrated that adequate UCE dating is consistent with dating from mitochondrial and nuclear sequences, and across studies with varying taxonomic representation.Divergence times within Cracidae from previous study were inferred from mitochondrial and nuclear loci, using the same UCE data in this study as backbone tree, as well as five same fossil calibrations . As expeThe divergence times for the Numididae and Odontophoridae were also similar between this study and previous multilocus studies , 48, whiLerwa pushed the time to the most recent common ancestor (TMRCA) of Erectile clade 2\u00a0Ma earlier, from 26.1 to 28.1\u00a0Ma; the inclusion of Tropicoperdix also pushed the TMRCA of Polyplectron clade significantly earlier  is a potentially surprising result; the same two accessions also exhibit a high degree of mitochondrial divergence [Overall, our divergence time estimates within Phasianidae were very similar to a recent study , e.g., tier Fig.\u00a0. Previouhip Fig.\u00a0. The deevergence , suggestWe generated a well-resolved galliform phylogeny that broadly sampled all genera, to allow robust estimates of divergence times, providing insights into the evolution of extant galliforms. Such phylogenies are important, as reconstruction of traits or ancestral ranges can be highly biased by phylogenies that are inaccurate or have biased taxon sampling , 31, 55,http://www.mycroarray.com/mybaits/mybaits-UCEs.html) and 100nt paired-end reads were generated using an Illumina  HiSeq 2500. We removed PCR duplicates from demultiplexed reads with PrinSeq-lite 0.20.4 [We sequenced one species from each galliform genus not represented in previous studies  [Struthio camelus), kiwi , and mallard (Anas platyrhynchos) from GenBank  to provide more distant outgroups to reduce the stochastic error in time estimation [https://phyluce.readthedocs.io/en/latest/index.html. We produced our data matrices using the standard PHYLUCE [We added these newly sequenced species to data from previous studies   and harv PHYLUCE  pipelineTo test the effects of different gene shopping schemes on divergence time estimation (see below), we used a subset of the taxa to reduce computation time, using a data matrix generated using PHYLUCE as described above. For these analyses, we selected 44 galliform species including all genera from Megapodiidae, Cracidae, Numididae, Odontophoridae, and eight genera from Phasianidae, representing all the three major clades identified within Phasianidae , as wellWe used RAxML 8.2.12  under a For the 48-taxon dataset, the ML tree from the 75% complete matrix was used as guide tree for divergence time estimation. However, we used the sequence data in the 95% and 100% complete matrix for divergence time estimation (see below).We conducted divergence time estimation using a total of seven schemes based on the 48-taxon dataset Fig.\u00a0. To miniTo avoid over-fitting models, phylogenetic analyses on large datasets frequently combine loci into larger partitions . MethodsWe investigated the parameters of our different schemes. For each locus included in one of the schemes, we obtained the aligned locus length, the GC content, and alpha parameter of the gamma distribution from RAxML analyses. We compared the locus lengths for each of the gene shopping schemes using the Independent-Samples Kruskal\u2013Wallis Test in SPSS 26. We also used the python script Alignment Assessment  to estimGallinuloides wyomingensis has led to overestimation of divergence times in Galliformes [For all the seven alignments from the 48-taxon dataset, we implemented MCMCTREE (PAML 4.9j ) with apliformes . Therefoliformes , 11, 31.liformes ). The paliformes . We ran liformes  to ensurUsing what our results suggested was an appropriate gene shopping scheme , we estimated divergence times of Galliformes on the ML topology from the 135-taxon dataset using the same methods described above.Additional file 1:\u00a0Figure S1. Species tree for the Galliformes from the 135-taxon dataset. Estimation was conducted on the 75% complete matrix using SVDquartets.Additional file 2:\u00a0Figure S2. ML phylogeny for the Galliformes from the 48-taxon dataset. Estimation was conducted on the 75% complete matrix using RaxML.Additional file 3:\u00a0Figure S3. Boxplot for the locus lengths for each of the gene shopping schemes.Additional file 4:\u00a0Table S1. UCE phylogenomic studies of Galliformes and novel UCEs used in this study.Additional file 5:\u00a0Table S2. Divergence time estimations (in 10 millions of years) of different gene shopping schemes for the 48-taxon dataset.Additional file 6: Table S3. Divergence time estimations (in 10 millions of years) from the most-tree like partition for the 95% complete of the 48 and 135-taxon datasets.Additional file 7:\u00a0Table S4. Fossils used to time-calibrated the galliform phylogeny."}
+{"text": "Partner notification (PN) is an essential element of sexually transmitted infection (STI) control. It enables identification, treatment and advice for sexual contacts who may benefit from additional preventive interventions such as HIV pre- and post-exposure prophylaxis. PN is most effective in reducing STI transmission when it reaches individuals who are most likely to have an STI and to engage in sexual behaviour that facilitates STI transmission, including having multiple and/or new sex partners. Outcomes of PN practice need to be measurable in order to inform standards. They need to address all five stages in the cascade of care: elicitation of partners, establishing contactable partners, notification, testing and treatment. In the United Kingdom, established outcome measures cover only the first three stages and do not take into account the type of sexual partnership. We report an evidence-based process to develop new PN outcomes and inform standards of care. We undertook a systematic literature review, evaluation of published information on types of sexual partnership and a modified Delphi process to reach consensus. We propose six new PN outcome measures at five stages of the cascade, including stratification by sex partnership type. Our framework for PN outcome measurement has potential to contribute in other domains, including Covid-19 contact tracing. Partner notification (PN) is a key strategy in sexually transmitted infection (STI) control. It entails identifying and informing sex partners of persons diagnosed with STI to facilitate their timely testing and treatment, to prevent reinfection of the person diagnosed (index patient) and onward transmission of infection . PartnerThe provision of PN, and monitoring of its performance, is key to improving the effectiveness of public health services for STI care. The process of PN can be described in a care cascade, with distinct stages and outcomes that can contribute to processes of service quality improvement. Standards used to monitor PN outcomes should address all steps in the PN cascade and should consider key factors that affect PN outcomes. In the United Kingdom (UK), the British Association for Sexual Health and HIV (BASHH) has published standards for PN for bacterial STI, with auditable outcome measures, last updated in 2012 . Figure Services for the diagnosis and care of sexually transmitted infections, including partner notification, vary across Europe. In the UK, sexual health clinics provide much of this care, especially in urban areas, with primary care services providing some less specialised testing and care. Also, the access to STI services varies across Europe, with gynaecology, primary care and sexual health services having a varying role, as do their approaches to partner notification. While legal and policy frameworks are diverse, the principles of partner notification in STI control remain the same, requiring different approaches to data collection and evaluation .In this Perspective article, we describe the process of evidence gathering, deliberation and consensus-building to address these gaps in current PN outcome measures and standards for bacterial STI in the UK, and to provide recommendations for future PN services internationally.Sexual behaviours, including the type of sexual partnership(s) and the force of infection in the sexual network, influence individuals\u2019 STI risk and their risk of onward STI transmission . PartnerCurrent PN outcome measures do not differentiate between different partner types. In the case of chlamydia infection, for example, the UK specifies as a standard that 0.6 contacts per index case with chlamydia should access sexual healthcare service within 4 weeks of the date of the first PN discussion. This standard is an average of outcomes for all sex partners, which does not take into account differences between sex partner types that affect STI prevention at the population level [Analysis of data from Britain\u2019s National Survey of Sexual Attitudes and Lifestyles has shown that sexual partnership type is associated with the probability of a recent STI diagnosis . This evA review and update of PN outcome measures and standards were commissioned by BASHH from the research team of the Limiting Undetected Sexually Transmitted Infections to RedUce Morbidity (LUSTRUM) programme. It is a 5-year programme of mixed methods research to improve PN methods, effectiveness and practice, with investigators from eight UK and one Swiss institution. The LUSTRUM study team includes experts in clinical sexual health medicine, health psychology, qualitative research, epidemiology, statistics, health economics and mathematical modelling and includes members of the National Surveys of Sexual Attitudes and Lifestyles  study team. The update was required to address PN outcomes which could be used to specify expected standards of care, and how these could be differentiated by sex partnership type.are given in the Supplement. We collated and reviewed all PN outcomes specified in PN guidelines in the following high-income English-speaking settings: Australasia , Canada, the UK and the United States.We undertook this update in three stages. Firstly, we performed two literature reviews to identify potential outcome measures for PN practice. We conducted a systematic review on MEDLINE building on a 2013 existing Cochrane review  and updaSecondly, we used a classification of sex partner types, developed by researchers in the LUSTRUM programme, drawing on qualitative research and existing literature on partnership type ,10. The Supplement. During this meeting, through a process of discussion and consensus building, a final set of optimal outcomes was chosen for final adoption into the BASHH PN national recommendations.Thirdly, we undertook a modified Delphi process to present the findings to experts, discuss the results and reach a consensus on measurable PN outcomes covering all five stages of the PN cascade and taking into account partnership type . In advaSupplement for the findings of this mapping exercise). These were circulated to the internal experts group in advance of their meeting. For each step in the PN cascade, we proposed a list of candidate outcomes as highlighted in orange in Finally, we mapped all PN outcome measures for bacterial STI identified in the review of RCT onto the PN cascade shown in The LUSTRUM team also developed five categories of sex partners drawing on empirical work within the research programme: 'committed, steady, occasional partner, new relationship and one-off partner'. It was highlighted that the LUSTRUM team had at that time demonstrated proof of feasibility by training staff members across 20 sexual health clinics to collect data using these partnership categories from index cases, subsequently collected in our RCT . ExpertsThe external experts concluded that overall, these five categories are useful and can potentially provide ways of reflecting on clinical practice and outcomes. They recommended that all PN outcome measures recommended should be stratified by partnership type. The external experts suggested a number of refinements to terminology, reflecting the complexities of clinical practice. These related to terminology for ongoing partnerships and how to determine when a previous partner becomes a new partner again.For effective evaluation of PN services for STI prevention, we need auditable outcome measures addressing each step of the PN cascade, which recognise the importance of partnership type. A more comprehensive and outcomes-focussed approach to auditing PN services will enable the cost-effective targeting of PN services and justify to funders the resources needed to do this. The need to establish and evaluate contact tracing processes at great scale and reach in the coronavirus disease (COVID-19) pandemic has sharply highlighted the need for meaningful and well understood PN outcome measures, which can be adapted to multiple infections and settings. The process of consensus established here in the context of bacterial STI, can contribute a framework for discussion well beyond the field of STI control.No ethical approval was required for this process of literature review and expert consensus building."}
+{"text": "In low-income countries, such as Mali, integrating mental health services into primary care is the most viable way of closing the treatment gap. This program aimed to provide a mental health training intervention to rural general practitioners (GPs), to organize community awareness activities, and to evaluate the impact on mental health knowledge and through the number of new patients diagnosed with mental disorders and managed by these general practitioners.a pre-test/post-test design and the monthly monitoring of the number of new patients diagnosed with mental disorders by the trained GPs were used to evaluate the effect of the training interventions  and of the community awareness activities.the mean knowledge score of the 19 GPs who completed the initial 12-day group training raised from 24.6/100 at baseline, to 61.5/100 after training (p<0.001), a 150% increase. Among them, sixteen completed the second 6-day group training with a mean score increasing from 50.2/100 to 70.1/100 (p<0.001), a 39.6% improvement. Between July 2018 and June 2020, 2,396 new patients were diagnosed with a mental disorder by the 19 GPs who took part in the program.despite limited data regarding the effect of the community awareness component at this stage, the findings from this study suggest that the training intervention improved GPs' knowledge and skills, resulting in a significant number of new patients being identified and managed. The worldwide prevalence of mental disorders is high: in a systematic review and meta-analysis of 174 studies (conducted in 26 high-income countries and 37 low-and middle-income countries (LMICs))  or 20.91As a result, GPs leave medical school with insufficient knowledge and negative attitudes towards mental disorders: a study conducted in India  showed tStudy design: this program involved a pre-test/post-test design to evaluate the effect of two face-to-face group mental health training workshops combined with individual follow-up supervision sessions, as well as the implementation of mental awareness activities directed towards the general population, and the monthly monitoring of the number of new patients with mental disorders diagnosed and managed by the trained GPs. Approval was received by the Direction Nationale de la Sant\u00e9, Ministry of Health, Republic of Mali in August 2017 to conduct these activities.Intervention: this 2-year intervention program which was implemented between June 2018 and June 2020, was based on three main groups of activities: capacity building, public awareness and advocacy, monitoring and evaluation.Capacity building: the aim was to leverage an existing network of rural GPs, by training 18 of them to diagnose and manage mental disorders, and to also train 8 of them so that they could, in turn, train other GPs. The initial face-to-face training was a 12-day session held in Bamako from 25 June until 7 July 2018. This initial training combined didactic presentations, questions and answers (Q and A) sessions, group workshops facilitated by experienced psychiatrists from the Point G University Hospital and from Martigues Hospital, France, as well as clinical hands- on- experience with two days spent at the Psychiatric Department of the Point G Hospital. The second training was a 6-day session which was held a year later (2-7 July 2019) also in Bamako. It included a practical part with four mornings of consulting sessions, with the support of the psychiatrists from the Psychiatric Department of the Point G Hospital and from Martigues Hospital, and a theoretical part in the afternoon with didactic presentations, Q and A session and sharing of the experience from the morning consultations. After each of these face-to face group trainings, individual follow-up supervision and mentoring sessions  were also organized at each GP health centre, during which, consultations were supervised, and medical records were discussed. These took place through February-March 2019, November-December 2019, and June 2020. In October 2019, eight of the trained GPs were also trained to become trainer of GPs and other health workers.Public awareness and advocacy: a workshop was organized in September-October 2018 with the participation of the National Center of Information Education and Communication for Health , psychiatrists from the Point G hospital, a representative of the Malian federation of traditional healers and herbalists (FEMATH), a representative of the national federation of community health associations (FENASCOM), a representative of the health communicators network and 3 GPs from the rural network. It led to the development of several educational tools: an educational flipchart with 11 key messages to be used by GPs and community health workers (CHWs) to inform, raise awareness and educate the local population as well as radio messages in 3 of the local languages . Between February 2019 and March 2020, there were radio broadcasts 4 times a week through 13 community radio stations, and 3 times a week through 2 regional stations. From May 2019, CHWs were trained to raise awareness, and advocacy meetings have been organized with traditional healers as well as religious and local leaders.Monitoring and evaluation: as the number of patients with mental disorders seen by the trained GPs was an important indicator to monitor, collecting monthly data was included as part of the activities to perform. From July 2018, the project manager has been following-up to ensure that every GP would send each month via whatsApp their report including the number of total new consultations, the number of new patients diagnosed with mental disorders as well as the number of these patients followed-up.Output and outcome measures: the change in knowledge has been measured through pre-and post-training questionnaires. For the 2018 training, the questionnaire included 40 questions with 0.5 point per question to get a total maximum score of 20, whereas in 2019, there were 20 questions with 1 point per question to get a total maximum score of 20. For the current analysis, these have been converted into a global score out of a maximum of 100. Beyond a global score, sub-scores were also calculated around specific disease areas and post-training scores improvement were expressed as percentage increases compared to pre-training scores. Other outcomes, such as the number of patients diagnosed with mental disorders and managed by the newly trained GPs, have been reported by each GP every month. They have been split into different diseases: psychoses, mood disorders, anxiety disorders, post-traumatic stress disorder (PTSD), addictions, autism and dementia. Other outputs such as the number of CHWs trained, number of people attending awareness meetings, number of traditional healers, religious and other local leaders met have also been reported on a monthly basis by GPs.Study area: this intervention program has been implemented across 19 GP community practices and rural clinics  in rural areas in six regions of Mali: Kayes, Koulikoro, S\u00e9gou, Sikasso, Mopti and Timbuktu.Statistical analysis: descriptive analysis has been performed for proportions, percentages, means and their corresponding standard deviations. Knowledge has been expressed as a score out of a maximum of 100 and the Wilcoxon-Mann-Whitney test for paired samples has been used to specifically examine changes between pre- and post-training. For all statistical analyses, p value of less than 0.05 has been set to determine statistical significance.Training and knowledge: nineteen GPs completed the initial face-to-face group training in June-July 2018. Their mean score increased from 24.6/100 before training, to 61.5/100 after training (p<0.001), i.e. a 150% increase compared to the pre-training score. As shown in Awareness and advocacy: between May 2019 and June 2020, 183 CHWs in the six targeted areas were equipped with educational flip-charts and trained to inform, raise awareness, educate communities regarding mental health and to address the many prejudices and the exclusion which people with mental disorders are often suffering from. Overall, approximately 76,000 people have been reached by awareness activities led by CHWs or GPs. In total, there were 336 community and regional radio broadcasts between February 2019 and June 2020, reaching an estimated 314,740 people in the six regions. Overall, GPs have met with fifty traditional healers and local leaders, mainly in Timbuktu (n=9) and in Mopti (n=10), to raise awareness about mental disorders and advocate for a better understanding of the diseases, their symptoms, and for greater collaboration with traditional healers. There have been some positive collaborative experiences, in particular regarding patients with mental disorders who have been referred by traditional healers to GPs in Gakoura, in S\u00e9guela (Kayes Region), in Markacoungo (Koulikoro Region), and in Koumantou (Sikasso Region). The most extensive collaboration has taken place in Markacoungo, particularly in Siratiguila, where the team came across a traditional healing centre, housing approximately 100 patients with various mental disorders, most of whom were being kept in inhumane conditions, chained up in a shed. To improve and humanize the care of these patients, an initiative involving a medical student and the traditional healer is being implemented and will be the topic of a thesis in medicine.New patients diagnosed: between July 2018 and June 2020, out of the 228,238 patient visits recorded by the 19 GPs who took part in the program, there were 2,396 new patients diagnosed with a mental disorder cumulatively  of the University of Sciences, techniques and Technologies of Bamako (USTTB) is a sign of the interest generated and of the relevance of the mental health training for GPs. It is also worth emphasizing that, over the two-year intervention period, the number of patients newly diagnosed each month remained relatively stable (mean of 100 patients per month \u00b1 20 SD)  confirming the inadequate training of GPs in mental health and the knowledge gap, which have been highlighted in other countries [\u00b1 20 SD) , despiteThe high percentage of psychoses diagnosed (42.0%) Despite some gaps in the evaluation of this program, it demonstrated positive effects on the knowledge and skills of GPs who were trained, and on the self-confidence of those GPs to diagnose and manage patients with mental disorders, as reflected by the number of new patients with mental disorders diagnosed and managed each month. Strategies to expand this program are currently being investigated, including a scaling-up leveraging the 8 GPs who have been trained to become trainers, combined with eLearning modules.Funding: financial support for this work was provided by Sanofi Global Health Programs.Mental disorders remain largely under-diagnosed and under-treated in Low and Middle-income Countries (LMICs), and in particular in sub-Saharan Africa, resulting in a very significant burden;In Mali, as in most LMICs, mental health is under-resourced, with very low specialized workforce, concentrated in Bamako, the capital city;In addition, primary healthcare professionals receive insufficient training to diagnose and manage people with mental disorders, making it difficult to integrate mental health services into primary care, as recommended by the World Health Organization.Our study suggests that a short mental health training intervention for rural general practitioners can improve GPs\u00b4 knowledge and skills, and result in a significant number of new patients being diagnosed and managed;Over a two-year period, the two face-to-face group training sessions on mental health, combined with individual follow-up supervision and mentoring sessions resulted in improved knowledge scores and improved skills. The effect on the number of patients newly diagnosed each month by the trained GPs remained sustained over the two-year intervention period."}
+{"text": "Patients with primary aldosteronism (PA) tend to exhibit a high prevalence of osteoporosis (OP) that may vary by whether PA is unilateral or bilateral, and responsive to PA treatment. To explore relationships between bone metabolism, PA subtypes, and treatment outcomes, we performed a systematic review and meta-analysis.The PubMed, Embase, and Cochrane databases were searched for clinical studies related to PA and bone metabolism markers. Articles that met the criteria were screened and included in the systematic review; the data were extracted after evaluating their quality. R software  was used for the meta-analysis.via sensitivity and subgroup analyses.A total of 28 articles were subjected to systematic review, of which 18 were included in the meta-analysis. We found that PA patients evidenced a lower serum calcium level , a higher urine calcium level , and a higher serum parathyroid hormone (PTH) level  than did essential hypertension (EH) subjects. After medical treatment or adrenal surgery, PA patients exhibited a markedly increased serum calcium level , a decreased urine calcium level , a decreased serum PTH level , and an increased serum 25-hydroxyvitamin D (25-OHD) level . The meta-analysis showed that the ser um PTH level of unilateral PA patients was significantly higher than that of bilateral PA patients  and the serum 25-OHD lower than that of bilateral PA patients . There were, however, no significant differences between PA and EH patients of 25-OHD, or BMD of femoral neck and lumbar spine. BMDs of the femoral neck or lumbar spine did not change significantly after treatment. The meta-analytical results were confirmed Excess aldosterone was associated with decreased serum calcium, elevated urinary calcium, and elevated PTH levels; these effects may be enhanced by low serum 25-OHD levels. The risks of OP and fracture might be elevated in PA patients, especially unilateral PA patients, but could be reduced after medical treatment or adrenal surgery. In view, however, of the lack of BMD changes, such hypothesis needs to be tested in further studies. Osteoporosis (OP) is a common disease characterized by decreased bone mass and strength, and destruction of bone microstructure, increasing the fracture susceptibility. OP can be divided into primary and secondary OP. As secondary OP is considered to be reversible, it is important to identify the cause thereof. Secondary OP can develop in those with diseases of the endocrine and blood systems, and of connective tissue; after drug use; and for many other reasons, of which the use of glucocorticoids , 2) is  is 2) isThe PubMed, Embase, and Cochrane databases were searched for clinical studies on PA and bone metabolism markers using the term \u201cHyperaldosteronism\u201d in combination with \u201cPTH, Calcium, BMD, Osteoporosis, and Secondary Hyperparathyroidism\u201d, with restrictions to the English language and human subjects; the databases were searched from their inceptions to March 6, 2022. We additionally searched the Grey Literature database. The above searches followed the PRISMA guidelines. All retrieved papers were imported into Zotero ver. 6.0.via adrenal venous sampling (AVS). All studies reported the levels of blood parathyroid hormone (PTH), serum calcium and urinary calcium, serum 25-hydroxy vitamin D (25-OHD), serum alkaline phosphatase (AKP), and bone-specific alkaline phosphatase (BAP); the bone mineral density (BMD); and the prevalence or morbidity from OP, fractures, and hyperparathyroidism (HPT).Inclusion criteria: Randomized controlled trials; cohort, case-control, or observational studies. A PA diagnosis verified by at least one confirmatory test ; or Exclusion criteria: The PA diagnosis was unclear or not specified. Clinical data were lacking. The experimental or case group, or cohort, were not PA patients. If PA subtypes were studied, there was no clear explanation as to how the subtypes were diagnosed.via discussion with, or arbitration by, a third specialist.Two researchers independently conducted literature screening and data extraction using a standardized form while operating in a double-blinded manner. We extracted the first author names and countries, type of study, publication year, subject populations, interventions, basic clinical characteristics, bone metabolism markers , and follow-up data after PA treatment. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of case-control and cohort studies. Any disagreement between the two researchers was settled 2 test, and considered that this was absent when I2 < 50%. If heterogeneity was absent, we used fixed-effect models for meta-analysis, but employed random-effect models when heterogeneity was in play and the causes thereof were not discernible. Publication bias was assessed using the Egger\u2019s test; we also drew funnel plots during meta-analyses of at least 10 studies. Visual confirmation of funnel plot symmetry, or an Egger\u2019s test P-value > 0.05, indicated no publication bias. We performed sensitivity analysis on only \u201chigh quality\u201d studies . We also performed subgroup analyses to explore the sources of heterogeneity.R software  (the \u201cmeta\u201d package ver. 5.2-0) was used for statistical analysis. We calculated mean differences (MDs) between patients and controls (with 95% confidence limits [CIs]) for continuous variables, and odds ratios (ORs) with 95% CIs for dichotomous variables. We sought heterogeneity using the Ivia AVS after ACTH stimulation; four via AVS without ACTH stimulation; and one via AVS both with and without ACTH stimulation); all presented the serum calcium and urinary calcium and PTH and 25-OHD levels; and the BMD.A total of 494 articles were retrieved, including 270 from PubMed, 215 from Embase, 9 from Cochrane, but none from the Grey Literature Figure\u00a01Of the 28 included articles, 26 were case-control studies, one a cohort study, and one a descriptive study. The quality of the case-control studies is shown in Eight studies reported significantly lower serum calcium levels in PA than EH patients . The urinary calcium level was significantly higher in seven studies that compared PA patients to EH subjects . Ten studies found that the serum PTH level was significantly higher in PA patients than in EH subjects . While no significant differences were found between PA and EH patients on 25-OHD, on BMD of femoral neck and lumbar spine and on AKP Figure\u00a02Nine studies reported significantly higher serum calcium levels  after than before treatment of PA patients. The urinary calcium level was significantly lower after treatment  in seven studies; the serum PTH level was significantly lower in 12 studies ; and the serum 25-OHD was significantly higher  in nine studies. None of the BAP level or the BMDs of the femoral neck or lumbar spine changed significantly after treatment Figure\u00a03The serum PTH level was significantly higher in unilateral than bilateral PA patients  in eight studies (nine datasets). Six studies found that the serum 25-OHD level was significantly lower in unilateral than bilateral PA patients . However, no significant differences were found in terms of the serum calcium or urinary calcium level or the BMDs of the femoral neck and lumbar spine Figure\u00a04Publication bias in terms of PTH levels was sought in works that compared PA patients to EH subjects and PA patients before and after treatment. The funnel plots were substantially symmetrical, and the Egger\u2019s test revealed no obvious publication bias Figure\u00a05The median NOS score was 8. Thus, we repeated the analysis including only \u201chigh quality\u201d studies (NOS \u2265 8). None of the AKP or BAP level, or the BMD, were included in sensitivity analysis because too few studies reported such data. After excluding \u201clow quality\u201d studies, the above data on urinary calcium and serum PTH levels were largely confirmed. In contrast, no significant differences were found between PA patients and EH subjects in terms of serum calcium levels, or between PA patients before and after treatment in terms of serum 25-OHD levels. However, the serum calcium level differed significantly between unilateral and bilateral PA patients Table\u00a02.All of study location (Asia or Europe), the assays used to measure serum PTH and 25-OHD, and the AVS procedure (with or without ACTH stimulation) might have affected our assessments; we thus performed three subgroup analyses. At least one such analysis eliminated the heterogeneity, except in terms of the serum calcium, PTH, and 25-OHD comparisons between PA patients and EH subjects Table\u00a03.We consistently found that PA patients  were at higher risk of OP than EH subjects but the risk was reduced after medical treatment or surgery. Thus, aldosterone may affect both PTH secretion and bone metabolism.Excess aldosterone directly affects bone formation and resorption; inhibition of the RAAS system reduces bone loss . The folResnick et\u00a0al.  were the2D receptor complex inhibits the expression of renin in vitro , . L. L2D recAfter excluding the effect of hypertension , PA patiThe trabecular bone score (TBS) is a new indicator of bone microstructure, used to evaluate bone quality and fracture risk. Several studies have suggested that aldosterone may trigger osteopenia by destroying bone microstructure rather than by reducing the BMD , 54); t; t54); tThe effect of aldosterone on BMD remains controversial , 23), a, a23), aThis possibility remains controversial , 17), , , 26), , , 15), , , 13, 19,There were 2 meta-analyses before our research. Loh et\u00a0al.  publisheExcess aldosterone was associated with decreased serum calcium, elevated urinary calcium, and elevated PTH levels; these effects may be enhanced by low serum 25-OHD levels. The risks of OP and fracture might be elevated in PA patients, especially unilateral PA patients, but could be reduced after medical treatment or adrenal surgery. The lack of BMD changes after treatment, indicates, however, that PTH and calcium changes may also represent an epiphenomenon of limited clinical significance, so more research is needed, either to confirm, or to refute the notion of any significant change in fracture rate in PA patients. TBS may be a better indicator than BMD when screening for OP in PA patients. More research is needed on how aldosterone may affect bone metabolism and the possible association between TBS and aldosterone.The original contributions presented in the study are included in the article/AW and ZL designed the study. HL and XH collected the data. AW performed the meta-analysis and drafted the manuscript. JL, HX, ZN and LZ partially planned the research. ZL edited the manuscript. All authors contributed to the article and have approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The chronic pain of patients with knee osteoarthritis (KOA) seriously affects their quality of life and leads to heavy social and economic burden. As a nondrug therapy in Traditional Chinese Medicine (TCM), Tuina is generally recognised as safe and effective for reducing the chronic pain of KOA. However, the underlying central mechanisms of Tuina for improving the pain of KOA are not fully understood.P<0.05 will be the statistical significance level.This study will be a randomised controlled trial with a parallel-group design. A total of 60 eligible participants will be assigned to the Tuina group or healthcare education group (Education group) at 1:1 ratio using stratified randomisation with gender and age as factors. The interventions of both groups will last for 30 min per session and be conducted twice each week for 12 weeks. This study will primarily focus on pain evaluation assessed by detecting the changes in brain grey matter (GM) structure, white matter (WM) structure, and the cerebral functional connectivity (FC) elicited by Tuina treatment, e.g., thalamus, hippocampus, anterior cingulate gyrus, S1, insula, and periaqueductal grey subregions (PAG). The two groups of patients will be evaluated by clinical assessments and multimodal magnetic resonance imaging (MRI) to observe the alterations in the GM, WM, and FC of participants at the baseline and the end of 6 and 12 weeks\u2019 treatment and still be evaluated by clinical assessments but not MRI for 48 weeks of follow-up. The visual analogue scale of current pain is the primary outcome. The Short-Form McGill Pain Questionnaire, Western Ontario and McMaster Universities Osteoarthritis Index, 36-Item Short Form Health Survey, Hamilton Depression Scale, and Hamilton Anxiety Scale will be used to evaluate the pain intensity, pain feeling, pain emotion, clinical symptoms, and quality of life, respectively. MRI assessments, clinical data evaluators, data managers, and statisticians will be blinded to the group allocation in the outcome evaluation procedure and data analysis to reduce the risk of bias. The repeated measures analysis of variance (2 groups \u00d7 6 time points ANOVA) will be used to analyse numerical variables of the clinical and neuroimaging data obtained in the study. The results of this randomised controlled trial with clinical assessments and multimodal MRI will help reveal the influence of Tuina treatment on the potential morphological changes in cortical and subcortical brain structures, the white matter integrity, and the functional activities and connectivity of brain regions of patients with KOA, which may provide scientific evidence for the clinical application of Tuina in the management of KOA.Chinese Clinical Trial Registry ChiCTR2000037966. Registered on Sep. 8, 2020.The results will be published in peer-reviewed journals and disseminated through the study\u2019s website, and conferences. Knee osteoarthritis (KOA) is an important cause of knee pain and disability, causing serious personal and social burden all over the world , 2. The The International Society for the Study of Pain redefines pain as an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage . KOA paiChronic pain of KOA is complex . The braHuangdi Neijing, which plays an important role in the health of Chinese people for thousands of years. It takes advantage of the hands of the therapist to manipulate the surface of the patient\u2019s body. The relaxation techniques in Tuina such as pressing and kneading exert significant analgaesic effect, improving mood, improving patients\u2019 confidence in prevention and treatment, promoting rehabilitation, and reducing the use of health services [Tuina is recorded in the classic work of ancient Chinese medicine services , 25. Tuiservices , regulatservices . Moreoveservices \u201334.An education programme such as a self-management programme can provide knowledge and skills that will come in handy in helping patients improve the management of their condition. Self-management skills are essential for treatment and are strongly recommended in the guideline for osteoarthritis management . For mosTuina is a promising treatment option for KOA . It is bHowever, studies on the longitudinal effect and central analgaesic mechanism of Tuina in patients with KOA chronic pain are few , 48\u201350. All patients with KOA will be recruited from the Tuina department of Yueyang Hospital of Integrated Traditional Chinese and Western medicine, Shanghai University of TCM. Recruitment methods will include posters, online advertisements, and leaflets. Written informed consents will be collected taken from all patients. A total of 60 patients with KOA chronic pain will be randomly assigned into a Tuina group and an Education group at 1:1 ratio.This study\u2019s protocol has been approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of TCM. The trial was also registered to the Chinese Clinical Trial Registry.This is a single-centre and parallel randomised controlled trial. A total of 60 participants diagnosed with KOA based on the American College of Rheumatology (ACR) criteria (2019 revised version)  will be Outcome measurements and MRI scans will be assessed at the baseline and at 6 and 12 weeks after treatment, followed by 48 weeks of clinical follow-up. The changes in clinical variables and cerebral activity of each group will be analysed after data collection.Evaluators and statisticians will be blinded to the group allocation during the procedure of outcome evaluation and data analysis , 53. TheMeet the diagnostic criteria for KOA set by the ACR in 2019 Are aged between 40 and 60 years, male or female, right-handed, with left knee painHave I\u2013II degree knee joint radiological change on the Kellgren\u2013Lawrence scaleHave an average knee pain score on a visual analogue scale (VAS) \u2265 3 (range from 0 to 10) in the past 3 monthsVolunteer to take part in the study and sign the informed consent formCan understand and follow the protocol.Mainly to ensure the feasibility of the clinical study and the better baseline balance of the two groups, and because the incidence rate of KOA patients aged 40\u201360 is relatively high , so it iAre taking analgaesics or anaesthetics in the past 1 month that may influence brain-imaging outcomesHave received any other treatment in the past 1 month, such as exercising therapy, pharmacological intervention, or operationAre pregnant or lactating womenAre suffering from mental disease, neurological disease, infectious disease, gastrointestinal disease, cardiovascular and cerebrovascular disease, immunologic disease, respiratory disease or kidney disease, any other chronic pain symptoms, or have a history of brain injury with loss of consciousnessAre diagnosed as cancer, tuberculosis, rheumatism or rheumatoid arthritis, gout, or joint traumaHave MRI contraindications such as claustrophobia, cardiac pacemaker, defibrillator, heart stent, or intrauterine deviceHave skin lesion around the knee jointAre unsuitable to continue to participate in the study owing to adverse events or serious adverse events during the trialAre unsuitable to continue to participate in the study because of serious deterioration of disease or some complications and special physiological changesUses concomitant medication during the 12-week treatment period is a suspension criteria. Non-compliance to concomitant interventions will be monitored by daily dairies and WeChat queries.Withdrew from the studyLoss of contact\u03c3 of the two groups is equal, i.e., \u03c3=2.1. If we set \u03b1=0.05 as the type I error, \u03b2=0.10 as the type II error, and 1\u2212\u03b2=0.90 as the power, the final result is that we need at least 25 samples in each group. For the primary outcome, VAS will be assessed at baseline and at the end of 6 and 12 weeks of treatment, followed by 12, 36, and 48 weeks of follow-up. The statistical significance is defined as P < 0.05, and the 95% confidence interval will be reported.Sample size is required to compare two group mean values. The calculation method of bilateral equality of two samples is adopted  for two Many MRI studies have reported statistical significance with 12 to 21 patients per group , 58\u201360. A total of 60 eligible participants will be assigned to the Tuina or Education groups at 1:1 ratio using stratified randomisation with gender and age as factors in this research. Stratified randomisation is achieved by performing a separate randomisation procedure within each of two subsets of participants (gender and age). First, participants will be grouped by gender. Then, male and female participants will be grouped by age. After grouping, there will be four subgroups: (1) men aged < 50, (2) men aged \u2265 50, (3) women aged < 50, and (4) women aged \u2265 50. In these four subgroups, the subjects will be randomly divided into the Tuina group and Education group. Finally, all Tuina groups and Education groups are merged to form a new Tuina group and Education group. This step ensures good balance amongst participant characteristics in each group and close balance amongst the numbers of participants receiving each intervention within each stratum. The created random number lists will be generated by a random number generator  and will be sent to the therapist with sequentially numbered, opaque, and sealed envelopes by an independent assistant. The therapist will open random-allocation envelopes and allocate the participants accordingly to the Tuina or Education group.Participants and therapists will not be blinded to treatment allocation, because of the features of the Tuina treatment and the education intervention. For participants, their subjective consciousness will affect the outcomes, which is unavoidable. Nevertheless, the evaluators, data managers, and statisticians will be blinded to the group allocation in the outcome evaluation procedure and data analysis throughout the entire procedure to reduce the risk of bias. The data will not be accessible to the evaluators until the collection is finished.The participants will receive a total of 24 treatments in 12 weeks. The interventionists, who hold a PhD, must have at least 10 years of experience with Tuina treatment and be skillful in healthcare education for the participants. They are also required to have passed a clinical test to ensure consistency of the trial. The clinical test primarily includes the time of Tuina, the positioning of the treatment site, and the application of specific Tuina manipulation types. The standard operating procedures primarily include Tuina\u2019s time, treatment site, and manipulation category, as well as the time and content of health education. In the process of Tuina or Education intervention, the two groups will not allow any concomitant medications or other interventions. However, concomitant medications or other interventions can be used during subsequent follow-up, and the treatment will be recorded in detail.The selected acupoints for the Tuina treatment include EX-LE02 (heding), EX-LE04 (neixiyan), EX-LE05 (xiyan), GB34 (yanglingquan), SP10 (xuehai), ST34 (liangqiu), ST32 (futu), BL40 (weizhong), BL57 (chengshan), GB31 (fengshi), and Ashi acupoints around the knee joint. An ashi point is a temporary acupoint where sensation of acid, distension, and pain exist, which can appear anywhere in knee joints .Referring to previous studies , 62\u201364, The Osteoarthritis Research Society International (OARSI) and the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) updated the guidelines on KOA in 2019 . In the The education intervention will be given to the participants with two sessions per week. The first health education session is a group meeting using a PowerPoint presentation, which will last 30 min. An educational booklet and a video will be provided to the patients to accomplish the home-based online e-learning module. The later sessions will be conducted as one-to-one communication focused on personal needs to guide patients to implement the key education in their lives. The programme includes two broad components: (1) introduce key concepts of pain biology, and (2) present specification of recovery.Each patient will be given an introduction about the underlying mechanisms, predisposing factors, and prognosis of KOA. Pain will be explained as the conscious part of the response, which can be influenced by many factors as a protective output . IntervePatients with KOA will be instructed not to take any caffeine , as well as other medications, weight-loss techniques, or physical activity to avoid confounding interference for the analgaesic effect of the intervention in the two groups. In cases of severe knee pain, ibuprofen (300 mg per capsule with sustained release) will be allowed as rescue medication and should be recorded on a knee-pain diary. After the end of the study, patients will be taught weight-loss techniques and physical activity. Patients are also asked to record the name, dose, date, frequency, and exact time of the medications used, as well as to report to the researcher if they take any medications during the study. Patient adherence will be maintained and monitored by good communication between the therapist and patient and by constant reminders.The participants\u2019 conditions will be carefully monitored by therapists during the process. If adverse events occur, participants with adverse events will stop participating in the trial. Immediate medical care will be provided to any participant experiencing adverse reactions that can lead to hospitalisation, for example fracture or coma. The details will be recorded in the case report forms (CRFs). The ethics committee will conduct an interim analysis and make a decision, which will be determined according to the severity of the adverse events of the participants. For example, conditions that lead to hospitalisation or even life-threatening are severer, whereas conditions such as muscle pain that can heal on their own are milder. Reports will be made to the ethics committee with statements and detailed reasons for trial suspension. The efficacy and safety of the intervention will be evaluated after 6 and 12 weeks of intervention and 48 weeks of subsequent follow-up. Evaluators can also be reformed with the clinical symptoms and AEs whenever the patients meet something important. After an adverse event is determined to be related to the trial plan, the research team will provide treatment costs and corresponding financial compensation for the damage inflicted by the trial plan.MRI data will be acquired using a 3.0-T magnetic resonance scanner  with a 32-channel phase-array head coil at the Medical Imaging Department of Yueyang Hospital of Traditional Chinese and Western Medicine, Shanghai University of TCM.On the pre-MRI evaluation procedure, the scanner will instruct the subject to rest for 10 min, relax, and keep calm. Throughout the entire scan, participants are asked to close their eyes, wear earplugs, keep their heads still throughout the scan, stay relaxed and awake, and not think of anything in particular.2, number of slices = 160, and slice thickness = 1 mm.Prior to the blood-oxygen-level-independent (BOLD) resting-state functional images, a high-resolution T1 image for each subject will be acquired with spin echo (SE) sequence, transverse sagittal scan, flip angle = 9\u00b0, pulse repetition time (TR) = 1900 ms, echo time (TE) = 2.93 ms, field of view (FOV) = 256\u00d7256 mm2, B-value2 = 1000 s/mm2, and 30 directions.DTI will be measured with the axial DTI mapping sequences, TR = 10,000 ms, TE = 89 ms, matrix = 240\u2009\u00d7\u2009240, slice thickness = 2 mm, B-value1 = 0 s/mm2, voxel size = 3.4 \u00d7 3.4 \u00d7 4.0 mm3, FA=90\u00b0, scanning time = 8 min 8 s, 240 volumes in total.The BOLD resting-state functional images will be obtained with an echo-planar imaging sequence as follows: coronal axial scan, 33 slices, 4 mm thickness, TE = 30 ms, TR = 2000 ms, FOV = 220 \u00d7 220 mmOn the post-MRI evaluation procedure, the subjects will be instructed to rest for 5 min. They may leave if there is no uncomfortable feeling.The MRI outcomes include the following: structure\u2013GM density, cortical thickness, subcortical nuclei volumes; diffusion\u2013FA and MD of WM integrity; fMRI\u2013functional connectivity (FC).http://surfer.nmr.mgh.harvard.edu). DTI data-processing pipeline will follow the diffusion toolbox in FSL to obtain FA and MD images, and voxelwise tract-based spatial statistics method will be applied to examine FA/MD changes. For the resting-state fMRI data, preprocessing and functional connectivity analyses will be performed with SPM12 software  by using MATLAB 2013b .The 3D T1 structure data analysis will be performed with FSL tools (FMRIB Software Library) . SIENAX The clinical outcome will be measured by six self-report questionnaires at baseline and the end of 6 and 12 weeks of treatment, followed by 12, 36, and 48 weeks of follow-up. The responses can reflect the degree of pain, sensation, emotion, and quality of life.The primary outcome measurement is VAS . VAS is The secondary outcomes are Short-Form McGill Pain Questionnaire (SF-MPQ) , WesternSF-MPQ is used to evaluate the pain, emotion, and psychological status of patients with KOA. It primarily comprises a 15-descriptor (11 sensory and 4 affective) rating on an intensity scale as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. A higher total score indicates more severe pain. The WOMAC is a self-administered questionnaire comprising 24 items divided into three subscales: pain (5 items), stiffness (2 items), and physical function (17 items). These measurements will be used to evaluate the symptom and quality of life improvement. A repeated longitudinal analysis will be used to evaluate the SF-MPQ mean scores.Furthermore, to investigate the influence of emotional state on the brain activity, HAMD  and HAMAMost items of the HAMD adopt the 5-level scoring method of 0\u20134 points. The standard of each level as follows: 0 = none; 1 = mild; 2 = moderate; 3 = severe; 4 = extremely severe. A few items adopt the 3-level scoring method of 0\u20132 points, and the grading standard as follows: 0 = none; 1 = mild to moderate; 2 = severe. The total score of HAMD is 78. A total score less than 8 means normal. A total score of 8\u201320 means possible mild depression. A total score of 20\u201335 means possible depression. A total score greater than 35 means severe depression. A repeated longitudinal analysis will be used to evaluate the HAMD mean scores.The total score of HAMA can better reflect the severity of anxiety symptoms. The total score can be used to evaluate the severity of anxiety symptoms and the effect of various drugs and psychological intervention in patients with anxiety and depression. According to the data provided by the China scale collaboration group, a total score \u226529 means possible serious anxiety, \u226521 means possible obvious anxiety, \u226514 means anxiety, \u22657 indicates possible anxiety, and <7 means no anxiety symptoms. A repeated longitudinal analysis will also be used to evaluate the HAMA mean scores.The screeners will collect data on the baseline characteristics when the patients are recruited. CRFs include observation and scanning time points, outcome measures, adverse events, and safety evaluations. Doctors are blinded to evaluate various clinical pain indicators and fill-in relevant information timely and accurately according to the CRF requirement. Only outcome assessors can access the CRFs and perform double-data entry. Then, two data administrators, who are beyond the research team and blinded to group allocation, will independently receive the completed CRF and enter them into an Excel database . They are required to have completed rigorous training for the data monitoring. Then, they will enter the real-time data into the Chinese Clinical Trial Registration Center, in which the electronic data-management system will be used to track and monitor the test data in real-time in the Department of Science and Technology in Yueyang Hospital. During the test, data administrators can access and manage data. After the test, all researchers will have access to the data, and non-researchers will not have access.Clinical data will be analysed through the intention-to-treat assumption. For all outcomes, we use longitudinal models where participant outcomes and treatments are collected at a multiple follow-up times. Moreover, participants who meet the suspension criteria will be removed from the trial, and the modified intention-to-treat analysis will be adopted. For the missing data, several sensitivity analyses will be conducted to evaluate their effects on the results of the experiment. All analyses were performed with SPSS 21.0 statistical software  by statisticians who are blinded to the group allocation.P < 0.05, and the 95% confidence interval will be reported.The baseline characteristics will be expressed with descriptive statistics for the two groups, which are reported as the mean \u00b1 standard deviation. A Kolmogorov\u2013Smirnov test with Lilliefors correction will be used to analyse all quantitative variables to determine whether they follow a normal distribution. Parametric statistics (Tukey test) or nonparametric statistics (Wilcoxon rank-sum test) will be used for the within- and between-group analyses in accordance with the results of the homogeneity and normality analyses. When the initial homogeneity and normality of data distribution are found, repeated-measure ANOVA will be implemented, or the Friedman test and Kruskal\u2013Wallis test will be used when initial homogeneity but non-normality of data distribution is found, or only linear mixed model will be adjusted for age and gender if the initial homogeneity is not found. Adverse events in each group will be documented as percentage for safety assessments by using the chi-square test or Fisher\u2019s exact test. Statistical significance is defined as During the processing of the trial, quality control will be conducted with the management of the steering committee. To ensure the consistency of methods, professional trial method and regular monitoring technique should be trained before the researchers participate in the trial. If the study protocol is modified or corrected, the steering committee and ethics committee should be informed. The steering committee and ethics committee will ensure that the trial process works as planned, such as monitoring recruitment, potential harms, Tuina intervention, Education intervention, and data quality.Chronic pain in KOA often seriously affects the patients\u2019 quality of life. Alleviating joint pain is an important part of KOA treatment recommendations in the international OARSI guidelines 2014)  and the 4  and thTuina belongs to the key noninvasive and nondrug therapies of TCM with distinctive characteristics , 85, whiMultimodal MRI is a cutting-edge technology in the study of pain brain-network mapping and analgaesia mechanism. It can explore the mechanism of pain and evaluate and predict pain. It is extensively used in brain science and pain research. Brain MRI is one of the most commonly used neuroimaging technologies in the study of central mechanism given its advantages of its high-quality spatial resolution, no radiation, rapid imaging velocity, non-invasion, and affordable price \u201389. The This clinical trial will study the multidimensional evaluation of KOA pain and evaluate the physical function, quality of life, mental status (the level of anxiety and depression), and influence of emotional state on brain activity. We will use a combination of clinical behaviour and multimodal MRI indicators such as brain-function indicators, brain-structure indicators , and brain-network properties to perform confirmatory and interactive Tuina analgaesia research, which can fill the knowledge gaps in brain pivot analgaesia research in Tuina discipline in this field.Furthermore, owing to the great difference between humans and animals, as well as the particularity of Tuina manipulation, verifying the central regulatory mechanism of Tuina intervention on KOA analgaesia in animal experiments is difficult because of too many uncontrollable factors. The application of multimodal MRI to Tuina discipline can clearly explore the brain central characteristics of KOA pain only in clinical trial, as well as comprehensively and deeply analyse the central analgaesic regulation mechanism of KOA patients with chronic pain after Tuina, which is a new trend in the future research of Tuina. We can also provide a reference experimental paradigm for the cross-study on the analgaesic effect of TCM nondrug therapy.This research has an inevitable limitation associated with the difficulty in controlling the methodology of blinding during the Tuina and healthcare education intervention, which cannot be blinded for the participants and therapists. Patients can know their group and anticipate the effect of the treatment in advance, which may affect their outcomes. For a study involving complex, multicomponent mind\u2013body therapy, identifying a feasible, useful, and valid sham comparison group remains challenging, with no well-accepted solution.This trial is recruiting patients now. Participant recruitment started in Sep. 2020 and is expected to end in Dec. 2022. The version of this protocol is the 1st version and the completion time is 8 Sep. 2020."}
+{"text": "To analyze the application efficiency of two tracers for sentinel lymph node mapping in patients with endometrial cancer.The records of endometrial cancer patients treated in our hospital from July 2019 to July 2021 were selected. Among them, 29 patients received methylene blue suspension injection and 33 patients received nano activated carbon suspension injection. The staining of sentinel lymph nodes was recorded and the application efficiency of two different tracers were analyzed.Total detection rate, average number of sentinel lymph nodes and bilateral detection rate of nano activated carbon suspension injection were significantly higher than those of methylene blue suspension injection (P<0.05). Detection accuracy, positive predictive value and sensitivity of nano activated carbon suspension injection were significantly higher than those of methylene blue suspension injection (P<0.05). Incidence of complications was the same in the two groups (P>0.05). Tracing time of nano activated carbon suspension injection was significantly lower than that of methylene blue suspension injection, and the total duration was significantly higher than that of methylene blue suspension injection (P<0.05).Nano activated carbon can obtain good detection effect in sentinel lymph node recognition in endometrial cancer patients, with shorter tracing time and higher total duration than methylene blue suspension. Endometrial cancer belongs to a group of epithelial malignant tumors, and occurs in the endometrium of patients, mostly postmenopausal and perimenopausal women. It occurs with the incidence of about 200 thousand new cases every year and is considered the third gynecological malignant tumor causing death after ovarian and cervical cancers,5The main treatment for the endometrial cancer is staged surgery, including abdominal paraaortic lymphadenectomy, pelvic lymphadenectomy, double appendectomy and total hysterectomy. Lymph node involvement is the most important diagnostic factor for the initiation of adjuvant therapy. Most patients with this disease are treated with early hysterectomy, simultaneous removal of bilateral accessories, sometimes combined with abdominal aorta and pelvic lymph node dissection. However, there is still no consensus on the unified treatment mode and scope.th). A total of 62 patients were collected in this study.Patients with endometrial cancer treated in our hospital from July 2019 to July 2021 were selected as the research object, and the patient\u2019s medical records were analyzed. The ethics committee of our hospital approved the study  based on the injected tracer. Tracer injection was performed following the same procedure for the patients. Briefly, patients were given general anesthesia, followed by routine disinfection and towel laying, routine exploration of the abdominal cavity and pelvic cavity, exposure of the cervix, and gauze pad application to protect the surrounding tissues and organs. For methylene blue suspension injection, 4ml of methylene blue suspension was selected as the tracer. For nano activated carbon suspension injection, patients were administered 3ml of nano activated carbon suspension. The anterior surface of the middle part of the cervix, the midpoint of the posterior wall and the midpoint of the anterior wall were selected as the injection sites, and each site was injected with 1ml of nano activated carbon suspension. The injection points were compressed and electrocoagulated to prevent dye leakage. After the injection, the patient\u2019s peritoneum was moved backward to expose the drainage areas of pelvic lymph nodes such as common iliac, abdominal aorta, internal iliac and inguinal. Stained lymph vessels were dissected along their direction, and the first stained lymph nodes were explored. The number and location of lymph nodes were recorded in detail. Pathological specimens of other lymph nodes and stained lymph nodes were obtained and sent for examination separately. The condition of uterus, peritoneum, aorta and uterine appendages were monitored, and lymph node dissection was carried out. After the operation, other lymph node specimens and sentinel lymph node specimens were sent to pathological examination.Evaluation of sentinel lymph nodes tracing was performed as follows: the number of cases detected in both hemi-pelvises and the total number of cases detected were recorded, and the detection rates were compared  \u00d7%). The following parameters were evaluated: average number of detected cases in patients was recorded; ii) effectiveness of tracing methods /total number of cases \u00d7%); iii) predictive value  \u00d7%); iv) sensitivity  \u00d7%); v) complications . Tracing time and total duration of the procedure was recorded.s) and t-test was performed. The counting data were expressed by n [%], and the test was done. P< 0.05 was considered statistically significant.SPSS 22.0 software was used for data analysis. The measurement data were expressed in  years, received methylene blue suspension injection. Thirty-three patients aged 23-59 years, with an average age of (51.03 \u00b1 7.71) years, received nano activated carbon suspension injection. There was no difference in the general characteristics of the two groups (P>0.05). Total detection rate, average number of sentinel lymph nodes and bilateral detection rate of nano activated carbon suspension injection method were significantly higher than those of methylene blue suspension injection (P<0.05) .The detection accuracy, positive predictive value and sensitivity of nano activated carbon suspension injection were significantly higher than those of methylene blue suspension injection (P<0.05) . The incTracing time of nano activated carbon suspension injection was significantly lower than that of methylene blue suspension injection, and the total duration was significantly higher than that of methylene blue suspension injection (P<0.05) .The results of this study showed that the total detection rate, average number of sentinel lymph nodes and bilateral detection rate, achieved with nano activated carbon suspension injection were significantly higher than those of methylene blue suspension injection (P<0.05). Nano activated carbon suspension injection resulted in significantly higher detection accuracy, positive predictive value and sensitivity as compared to methylene blue suspension injection (P<0.05). The incidence of complications was similar in both groups (P>0.05), while the tracing time of nano activated carbon suspension injection was significantly lower than that of methylene blue suspension injection. At the same time, total duration was significantly higher than that of methylene blue suspension injection (P<0.05). Li Y et al.At present, the commonly used sentinel lymph node detection methods in clinic mainly include three types: radionuclide tracing method, dye method and combined test method. Among them, radionuclide tracing method is associated with a high cost, and relevant imaging equipment and detectors need to be used in the test process. The actual operation of combined test method is complex and requires special instruments for auxiliary inspection. Compared with single tracer method, the cost of the combined test method is higher. Therefore, at this stage, fluorescent dye method is considered a key research topic. Compared with the traditional blue dye test, this test method can significantly improve the bilateral detection rate of sentinel lymph nodes. However, this test method also needs special imaging equipment and fluorescent imaging technology.He J et al. conducted a study to evaluate whether the use of methylene blue or activated carbon nanoparticles as tracers can increase the number of lymph nodes detected in systematic lymph node dissection tissues in video-assisted thoracoscopic surgery (VATS) for non-small cell lung cancer.Total detection rates of the two tracers for sentinel lymph nodes are similar, but due to the small sample size of this study and other factors, our results must be taken with caution. To further verify the accuracy of the test results, it is necessary to expand the sample size and to conduct further studies on a large sample size.Nano activated carbon can obtain good detection effect in the mapping and tracing of sentinel lymph nodes in patients with endometrial cancer. It shows shorter tracing time and longer total duration compared to methylene blue tracer. This study provides basis for more effective and comprehensive approach to clinical treatment of patients with endometrial cancer.WZ: Conceived and designed the study.YS, DN, ZW and SY: Collected the data and performed the analysis.WZ: Was involved in the writing of the manuscript and is responsible for the integrity of the study.All authors have read and approved the final manuscript."}
+{"text": "The use of ceftazidime (CAZ) with AVI is approved for several indications. This study evaluated the Eba and 23051 Pae non-duplicate clinically significant isolates, including 14216 Eba and 3002 Pae isolated from bloodstream infections, were collected in 56 countries in Europe, Latin America, Asia/Pacific (excluding mainland China), and the Middle East/Africa region. Susceptibility testing was performed by CLSI broth microdilution. Meropenem-nonsusceptible (MEM-NS) Eba and Pae isolates were screened for the presence of \u03b2-lactamase genes. Only 25% of MEM-NS Pae collected in 2020 were screened.A total of 67326 Eba and 90.4% Pae were susceptible to CAZ-AVI , more than any comparator tested (table). This was true of blood isolates as well, with 96.9% (Eba) and 90.4% (Pae) isolates susceptible to CAZ-AVI. CAZ-AVI was active against 89.9% of Eba collected from blood that were CAZ-nonsusceptible (NS), more than the comparators tested. While this was not true of CAZ-NS Pae blood isolates (52.9% susceptible to CAZ-AVI), the next most active comparator, amikacin, was active against only 3.9 percentage points more isolates. More MEM-NS isolates that screened negative for MBLs were susceptible to CAZ-AVI than any comparator tested (97.8% of Eba and 80.1% of Pae).Of all isolates collected, 97.3% Eba and Pae, except those carrying MBLs.CAZ-AVI provides a valuable therapeutic option for treating bloodstream infections caused by All Authors: No reported disclosures."}
+{"text": "Are there differences in treatment and clinical outcomes of patients hospitalized with COVID-19 associated with race?In this cohort study that included 43\u2009222 adult veterans hospitalized with COVID-19, Black veterans had lower odds of receiving COVID-19\u2013specific treatments, including steroids, immunomodulators, and antivirals.These findings suggest that variation in treatment contributes to differences in COVID-19 care between Black and White patients. This cohort study assesses within- and between-hospital variance in COVID-19 treatment and outcomes associated with race among hospitalized US veterans. Patients from racially and ethnically minoritized populations, such as Black and Hispanic patients, may be less likely to receive evidence-based COVID-19 treatments than White patients, contributing to adverse clinical outcomes.To determine whether clinical treatments and outcomes among patients hospitalized with COVID-19 were associated with race.This retrospective cohort study was conducted in 130 Department of Veterans Affairs Medical Centers (VAMCs) between March 1, 2020, and February 28, 2022, with a 60-day follow-up period until May 1, 2022. Participants included veterans hospitalized with COVID-19. Data were analyzed from May 6 to June 2, 2022.Self-reported race.Clinical care processes  were quantified. Clinical outcomes of interest included in-hospital mortality, 60-day mortality, and 30-day readmissions. Outcomes were assessed with multivariable random effects logistic regression models to estimate the association of race with outcomes not attributable to known mediators, such as socioeconomic status and age, while adjusting for potential confounding between outcomes and mediators.P\u2009=\u2009.17). Two-thirds of patients treated with supplemental oxygen or noninvasive or invasive mechanical ventilation also received systemic steroids, but Black veterans were less likely to receive steroids . Similarly, Black patients were less likely to receive remdesivir  or treatment with immunomodulatory drugs . After adjusting for patient demographic characteristics, chronic health conditions, severity of acute illness, and receipt of COVID-19\u2013specific treatments, there was no association of Black race with hospital mortality  or 30-day readmission .A total of 43\u2009222 veterans  with a median (IQR) age of 71 (62-77) years who were hospitalized with SARS-CoV-2 infection were included. Controlling for site of treatment, Black patients were equally likely to be admitted to the ICU (4806 Black patients [39.6%] vs 13\u2009427 White patients [43.2%]; within-center adjusted odds ratio [aOR], 0.95; 95% CI, 0.88-1.02; These findings suggest that Black veterans hospitalized with COVID-19 were less likely to be treated with evidence-based COVID-19 treatments, including systemic steroids, remdesivir, and immunomodulatory drugs. A 2022 study by Wiltz et al19 suggested that minoritized patients are less likely to receive outpatient treatment with monoclonal antibodies and inpatient treatment with remdesivir and dexamethasone. However, Wiltz et al19 did not account for differences in patient demographics, underlying health conditions, and clustering at the hospital level.Preexisting inequities in employment, income, housing, and health care access and differences in exposure risk and comorbidity burden have placed minoritized racial and ethnic populations, such as Black and Hispanic individuals, at greater risk for contracting COVID-19 and associated morbidity and mortality.In this study, we used a national cohort of veterans with granular clinical data to characterize differences in the treatment and outcomes associated with race among patients hospitalized with COVID-19. We categorized the pandemic into distinct periods based on waves of COVID-19 hospitalizations. We used statistical methods to account for differences in demographic characteristics, chronic health conditions, acute illness severity, outpatient treatments, and care location and performed sensitivity analyses using a 1:1 matched cohort. We hypothesized that processes of care and short- and long-term outcomes would differ by racial group and over time during the SARS-CoV-2 pandemic.STROBE) reporting guideline.This cohort study was approved by the VA Pittsburgh Healthcare System institutional review board with a waiver of the informed consent requirement per VA Common Rule, Title 38 CFR Part 16. This study is reported following the Strengthening the Reporting of Observational Studies in Epidemiology  health care system, the largest integrated health services system in the United States. In response to the SARS-CoV-2 pandemic, the Department of Veterans Affairs (VA) created the VA COVID-19 Shared Data Resource, as a regularly updated database with extensive demographic, clinical, pharmacologic, laboratory, vital signs, and clinical outcomes information derived from multiple validated sources.23 By limiting our cohort to veterans with at least 2 primary care visits in the 18 months preceding vaccine roll-out, we attempted to identify veterans who regularly use the VHA for their health care needs. Comorbidities were defined per the VA COVID-19 Shared Data Resource. We used the Charlson Comorbidity Index (CCI) to quantify preexisting comorbidities .25 The Area Deprivation Index (ADI) was used as a marker of socioeconomic deprivation .26 We categorized self-reported race into Black and White and ethnicity within race into Hispanic and non-Hispanic.We performed a retrospective analysis of veterans hospitalized with SARS-CoV-2 infection confirmed via laboratory polymerase chain reaction (PCR) testing at 130 VA hospitals between March 1, 2020, and February 28, 2022, with follow-up until May 1, 2022. To compare care processes and outcomes over time, we categorized hospitalizations during the pandemic into 5 distinct intervals  based on peaks and troughs of COVID-19\u2013related hospitalizations and in a more granular fashion by month of hospitalization . The firWe quantified clinical care processes, such as intensive care unit (ICU) admission; organ support measures, including invasive and noninvasive mechanical ventilation; and prone position therapy. We compared time from admission to initiation of mechanical ventilation and administration of COVID-19\u2013specific anti-inflammatory treatments between Black and White veterans. We included systemic steroids (dexamethasone and hydrocortisone), antivirals (remdesivir), and immunomodulators  in our analyses. Clinical outcomes of interest included in-hospital mortality, 60-day mortality, and 30-day readmissions, including common readmission diagnoses.2 tests as indicated. We constructed multivariable generalized linear mixed models to estimate the odds of receiving COVID-19\u2013specific treatments, invasive and noninvasive mechanical ventilation, and experiencing in-hospital death, death within 60 days, and 30-day readmissions. We used a directed acyclic graph  measures the association of the exposure variable race and outcome variable within a given hospital, whereas the between-hospital aOR reflects the association between the proportion of Black patients and the outcome of interest by comparing a hypothetical hospital that exclusively serves Black patients with a hospital that serves no Black patients.6We used descriptive statistics to summarize baseline characteristics and present these as medians with IQRs, means with SDs, and proportions, as appropriate. We compared differences in care processes and outcomes between racial groups and time periods using Wilcoxon rank-sum tests, Kruskal-Wallis 1-way analysis of variance, and \u03c7We performed sensitivity analyses and compared the same processes of care and clinical outcomes using a 1:1 matched cohort. Using random coarsened exact matching, we matched White veterans to Black veterans on age (10-year age categories), sex, ADI, CCI category, preexisting chronic kidney and liver disease, ICU admission, mechanical ventilation, oxygen requirement, vaccination status, week of hospitalization, and VHA facility.We tested the hypotheses that care processes and clinical outcomes varied by race within and between hospitals. We assessed the statistical significance of the differences within and between hospitals from each model by calculating the asymptotic variance of the difference and the corresponding 95% CI. We report within- and between-hospital differencesP\u2009<\u2009.05 was considered statistically significant. We did not adjust for multiple comparisons. We performed all analyses using R statistical software version 4.1.5 . Data were analyzed May 6 to June 2, 2022.A 2-sided We identified 43\u2009222 veterans  with PCR-confirmed SARS-CoV-2 infection hospitalized between March 1, 2020, and February 28, 2022. The median (IQR) age was 71 (62-77) years, and 2955 patients (8.9%) identified Hispanic ethnicity .P\u2009<\u2009.001) and varied by pandemic period, with the youngest patients hospitalized during periods 2 and 4 (P\u2009<\u2009.001). Black and White patients had a similar burden of preexisting health conditions , and the CCI decreased throughout the pandemic . Individual comorbidities varied by racial category. For example, coronary artery and chronic obstructive pulmonary disease were more prevalent in White veterans, whereas diabetes, hypertension, and chronic kidney disease were more prevalent among Black patients  were hospitalized. Most patients (38\u2009353 patients [88.7%]) were hospitalized during the latter 3 periods, which included the Delta- and Omicron-predominant periods . Most patients hospitalized during period 1 were Black; however, the proportion of White patients increased over time . The med 2 and 4 . Black ppatients . Most papatients .P\u2009=\u2009.17) (P\u2009=\u2009.04). Conversely, Black veterans were less likely to receive noninvasive ventilation , and it was less likely in hospitals serving a higher proportion of Black veterans . The duration of mechanical ventilation, time from admission to mechanical ventilation, and use of prone positioning were similar between racial groups, but proning was much less likely among hospitals serving more Black veterans . The proportion of patients treated with invasive mechanical ventilation was highest during the first period (322 patients [20.1%]) and lowest during the last period   . HoweverP\u2009<\u2009.001) (P\u2009=\u2009.004), as were patients admitted to hospitals treating higher proportions of Black patients  (P\u2009<\u2009.001), as were patients admitted to hospitals treating higher proportions of Black patients  (P\u2009<\u2009.001).Approximately two-thirds of patients (21\u2009022 patients [67.8%]) treated with supplemental oxygen or noninvasive or invasive mechanical ventilation also received systemic steroids. The proportion of patients treated with steroids among those treated with supplemental oxygen increased over time  . A more P\u2009=\u2009.03) . SimilarP\u2009=\u2009.02) . TreatmeTo understand whether hospital characteristics differed between better and poorer performing hospitals, we plotted the distribution of the within-center odds ratios for steroid treatment by race . After adjusting for patient demographic characteristics, chronic health conditions, severity of acute illness, and receipt of COVID-19\u2013specific treatments, there were no differences in hospital mortality between Black and White veterans , whereas 60-day mortality was lower for Black veterans   and declined significantly over time . During 78-0.94) .Of 38\u2009782 patients discharged alive, 5597 (14.4%) were readmitted within 30 days. The hospital readmission rate did not differ substantially between early and later pandemic periods (eTable 3 in the P\u2009=\u2009.04) or antiviral treatment with remdesivir , and the proportion of patients treated increased over time. For example, the 181 patients (38.7%) needed supplemental oxygen and were treated with steroids during periods 1 and 2, and nearly twice as many patients needed supplemental oxygen and received steroids during periods 3 to 5 (2458 patients [68.4%]). The proportions of patients treated by race and time period are summarized in eTable 4 in the P\u2009=\u2009.04). The proportion of hospital survivors readmitted by day 30 did not differ by racial group in the matched cohort.Our 1:1 matched validation cohort included 4062 Black and White veterans  and did not account for clustering within hospitals. Black patients in our cohort were less likely to receive remdesivir than White patients. Our results contradict the assumptions that differences in inpatient treatment by race and ethnicity may be due to differences in clinical indications for medication use based on age and prevalence of specific comorbidities, such as chronic kidney or liver disease, or reflect varying prescribing practices, protocols, and drug access by institutions that serve populations of differential racial and ethnic distributions.19 First, the VA issued a COVID-19 response plan,37 including specific treatment guidelines and distribution plans, applicable across the entire VA health care system. Second, our models accounted for clustering at the hospital level and preexisting chronic health conditions, including chronic kidney and liver disease. We derived within- and between-center aORs to measure the association between race and outcome variables to account for differences in the proportion of Black patients treated. Third, we performed sensitivity analyses that confirmed our results in a 1:1 matched cohort of Black and White patients matched on demographic characteristics, chronic health conditions, month of hospital admission, vaccination status, ADI, geography, and the severity of COVID-19.Our results confirm recent data from 41 US health care systems participating in the National Patient-Centered Clinical Research Network (PCORNet), which found lower monoclonal antibody treatment use among Asian, Black, and Hispanic patients and patients identifying as another race  SARS-CoV-2 infection compared with non-Hispanic and White patients.38 Using data from 27 US states, Gangopadhyaya38 reported significantly worse patient care for Black vs White patients treated in the same hospitals based on 6 of 11 safety indicators. Despite adjusting for patient characteristics, including insurance coverage type, these differences persisted and were not explained by hospitals that treated the largest proportion of minoritized patients.38 These findings are consistent with our results and suggest that differences in the quality of care are due to both within and between hospital differences. We compared hospital characteristics of hospitals that were in the bottom 20% vs the top 20% of the within-center aOR for steroid treatment among Black vs White patients. Hospitals in the bottom 20% were distinctly different from top-performing hospitals regarding size, complexity level, and their association with other health care quality metrics. These quality metrics were also used in a recent study that demonstrated a poorer quality of care for hospitals with higher monthly COVID-19 discharges and hospital size,12 suggesting that these routinely collected metrics may serve as a potential surrogate for other care processes in these hospitals.Within-hospital racial differences in the quality of hospital care have been described using various general and surgical patient safety indicators that measure adverse event rates of hospital-acquired illnesses or injuries in a 2021 report by Gangopadhyaya.We did not observe consistent differences in clinical outcomes between Black and White patients. Multiple factors influence clinical outcomes in addition to medical treatments, several of which were unavailable to us , all of which could have resulted in residual confounding. In addition, heterogeneity of treatment effect of COVID-19\u2013specific treatments may contribute to the lack of a robust association with survival outcomes. Identifying distinct patient populations who benefit from specific treatments may decrease the variation in care within and between hospitals.20 We adjusted for potential confounding between outcomes and the plausible mediators of the effect of race on outcomes, accounted for clustering within hospitals, and performed additional sensitivity analyses using a 1:1 matched cohort.Our study has several strengths, including data from a validated and well-maintained source and a large sample size of patients from 130 hospitals across the US, all within a single integrated health care system.Our study has several limitations. First, we performed retrospective analyses utilizing electronic medical record data subject to residual confounding. Second, we did not adjust for hospital bed capacity and subsequent associations with clinical care, patient preferences , treatment availability in the setting of demand-supply mismatch, and clinical outcomes. Additionally, our cohort consists of a predominantly older, male veteran population in the US, and our results may have limited generalizability to women, younger populations, and nations with different socioeconomic makeup than the USIn our cohort study, Black race was associated with lower odds of receipt of evidence-based COVID-19 treatments, including systemic steroids, remdesivir, and immunomodulatory drugs. Differences in care were partially explained by within- and between-hospital differences and underscore the need for a comprehension approach to minimize racial variation in COVID-19 care."}
+{"text": "Bentonites, as a clay mineral, serve in pig farms as adsorbents of toxic substances. They are mainly used to reduce the negative impact of mycotoxins to maintain the performance and health status of animals. The new genotypes of pigs are highly sensitive to a range of antinutrients, including mycotoxins. Currently, attention is focused on more effective adsorbents of mycotoxins with a higher adsorption capacity. Such materials are in great demand among feed manufacturers. However, there is a concern that these new materials may also adsorb too many essential nutrients and decrease animal performance. The aim of the experiment was to evaluate the effect of the new generation of purified bentonites on the efficiency and health status of the pigs.Forty-eight slaughtered pigs with an average weight of 31.2\u2009\u00b1\u20092.6\u00a0kg were included in the experiment. The pigs were divided into two groups (2\u2009\u00d7\u200924 pigs). Pigs were slaughtered at an average weight of 66.3\u2009\u00b1\u20095.2. The first group had a diet without clay (control\u2014C). The second group (treatment\u2014T) was fed a diet with a clay additive (purified bentonite) of 1.5\u00a0kg/t. Animals were fed the experimental diet for 35\u00a0days. In group T, a higher daily weight gain (by 4.8%) and feed intake (by 2.9%) was observed while the feed conversion decreased by 1.9%. There were no significant differences between the groups of pigs during observation in the evaluation of hematological, biochemical parameters of the blood. Morpho-pathological analysis of the jejunum showed similar signs of moderate lymphoplasmacytic infiltrate in the mucosa in the groups C and T, contained similar number of goblet cells.Taken together, the addition of the new generation of bentonite clays did not negatively influence the health status and the performance of pigs. Bentonite is a mineral authorized in Europe as a feed additive for reducing mycotoxin contamination in feed (EU Reg. No. 1060/2013) .Clays have been a standard ingredient in pig feed mixtures for several decades. In particular, bentonites, kaolins and zeolites hold the major share of the global market . Due to Adding clays to pig feed mixtures can potentially lead to a decrease in animal performance or damage to health. High adsorption capacity is characteristic particularly for bentonites. This property is mainly due to a very fine structure and a large adsorbent surface . AccordiBentonites can adsorb large organic molecules, polymer substances, complex ions, enzymes or ammonia. Thus, mineral nutrients such as zinc, manganese, selenium, cobalt, etc. can also be bound, with negative effects. Another potential risk of clay adsorbents is their toxicity, which is linked to the extraction site. If clays are obtained with natural or anthropogenic toxic compounds, such a product becomes potentially dangerous to the health of the pigs .Recently, there has been a trend of a steady increase in the adsorption capacity of bentonites used in animal nutrition. This effect can be achieved via advanced technological processes involved in grinding, purification and filtration. One goal of the scientific community should be to verify the safety of these processed bentonites for the organism health of animals. . PurifieIn assessing the pig\u2019s growth performance, no significant differences were observed between each stage of the experiment. The group with the addition of clay adsorbent showed a higher feed intake by 0.06\u00a0kg/day during the entire experiment. The feed conversion was lower by 0.04\u00a0kg and the daily gain was higher by 0.05\u00a0kg/day also in group T. The performance of the individual indicators during the experiment can be seen in Tables Liver and kidney weight was related to the body weight of pigs. From Table There were no significant differences between the control group and the treated group in assessing biochemical hematological and antioxidant parameters. The individual parameters corresponded to the physiological levels for the animal species. The results we can see in Table In group C, a lymphoplasmic inflammatory infiltrate in the mucosa, an abundance of eosinophils and isolated macrophages were observed. Lymphangiectasia of some villi and lymphatic vessels in the submucosal layer were found in some of the biopsies. The average number of goblet cells was 49 (counted in the HPF). Figure\u00a0Some authors are concerned that the use of new materials with higher adsorption capacity, such as functionalized bentonites by acid-activation, will lead to a decrease in animal performance . Other sThe improved growth performance of fattening pigs after the addition of silicate clay was also investigated in another study by Li et al. The authors explained this effect by the fact that clays can improve the integrity of the intestinal wall and positively influence the intestinal microbiome . After tIn our experiment, we found a similar trend. For group T, the daily weight increment was insignificantly higher by 5.1% and the feed conversion improved by 1.9% over the duration of the experiment. Other studies also indicate that the addition of clay (montmorillonite) to the diet of weaned pigs at doses of 0\u20135% did not affect the animal\u2019s performance. In contrast, feed intake decreased linearly with increasing doses .In our follow-up, the addition of purified bentonites had an inconclusive but positive effect on feed intake (an increase of 2.9%). The results of studies on the effect of clay substances on the diet of animals are inconsistent and the values vary from one observation to another. According to Alexopoulos et al.  this effA dose of 1 or 2% bentonite in the diet of weaned piglets did not significantly affect the performance of the animals as in our study . From thIn assessing the biochemical parameters in the pigs\u2019 blood, an evidential decrease in urea was observed in the experimental group of 1.98\u00a0mmol/L. We monitored the values of urea in the blood of pigs to a higher _____ in the control group\u20144.22\u00a0mmol/L, the experimental group had 5.45\u00a0mmol/l. The differences between our values and the values of Bederska-Lojewska and Pieszka (2019) are likely to be due to the different protein representations in the pigs\u2019 diet. Further evidence-based changes were observed in AST. This enzyme was shown to be higher in the group of animals supplemented with clays . The resTrckova et al.  have beeAfter overcoming mild infection, the following late-phase inflammatory responses were observed: minor destructions and desquamations of the intestinal epithelium with enterocyte shedding into the gut lumen, hyperaemic areas and areas with intestinal crypt hyperplasia. No marked differences between control and bentonite piglets were observed.The authors Trckova et al. stated that mild signs of inflammation were observed in the control group and experimental group fed by kaolin enriched feed . These rPigs have become sensitive to the negative effects of the environment. The goal of nutrition experts is to develop new materials that can eliminate the effects of toxic substances on the body . Thereby, evolutionary materials appear, which are characterized by a larger surface area and a higher adsorption capacity. Due to high adsorption capacity and large surface area, there are concerns about their enhanced adsorption of essential nutrients and decrease the performance of pigs. In our experiment, no adverse effect on the performance of fattened pigs, or biochemical and hematological parameters of blood was observed. There was also no difference between the control group and the experimental group of pigs during assessment the morpho-pathology of the small intestine and liver. Based on our results, we can conclude that purified bentonites do not pose a risk to fattening pigs. However, it would be advisable to test the results experimentally on more sensitive categories of pigs.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of the Expert Commission for Ensuring the Welfare of Experimental Animals of Mendel University in Brno . A total of 48 experimental pigs\u2014castrated males (DanBred) with an average weight of 31.2\u2009\u00b1\u20092.6\u00a0kg were housed in 12 identical pens measuring 2.43\u2009\u00d7\u20091.46\u00a0m. The animals were exposed to artificial lighting sources with a light intensity of 45\u00a0lx in the mode 12\u00a0h-light and 12-h dark cycle. According to the pre-fattening pigs' requirements, the microclimatic conditions of pigs' housing were maintained by an artificial computer-controlled ventilation system. The basic microclimatic standards were maintained following the requirements of the Act on the protection of animals against cruelty (No. 246/1992 Coll.). The composition and nutritional values of the feed rations are shown in Tables\u00a0All experimental animals were provided ad libitum access to feed and drinking water. In addition, the unconsumed feed mixture was monitored.2, average particle size 30\u00a0\u00b5m, low polarity, pH does not affect adsorption efficiency due to a high cation exchange capacity; dosing was chosen according to the manual) and served to test the mycotoxin adsorbent's potential adverse effect. Clay adsorbent was based on purified and activated bentonite . The first group had a diet without bentonite addition (control\u2014C). The second group (treatment\u2014T) was fed a diet with a purified bentonite of 1.5\u00a0kg/ton . The animals were left to rest and fast for about two hours before slaughtering. Then, they were slaughtered by electrical stunning  and exsanguination. In each slaughtered pig, kidney and liver weight were determined as percentages of the pigs' live weight. Blood samples were collected via the jugular vein into EDTA tubes and 10\u00a0mL heparinized vacuum, and then aliquots were centrifuged at 3000\u00d7g for 10 to collect plasma which was frozen at \u2212\u200920\u00a0\u00b0C until analysis. Liver and intestine samples were collected, washed in PBS buffer, and placed in 10% formalin until evaluation. Middle jejunum  samples were collected and frozen at \u2212\u200920\u00a0\u00b0C until analysis.For documentation of the activated bentonite structure Fig.\u00a0B, a scanAll feed samples were oven-dried at a temperature of up to 50\u00a0\u00b0C, then ground with a grinder to a particle size of 1\u00a0mm and analyzed for the basic nutrient content. The fiber was analyzed on an A200 Fiber Analyzer , nitrogenous substances according to the Kjeldahl method (N\u2009\u00d7\u20096.25), fat (by direct extraction according to Soxhlet method). The ash was analyzed using a calorimeter  after burning for 4.5\u00a0h in an oven at 550\u00a0\u00b0C. Energy .The method for AAA was adopted from Husek and Sweeley et al. In brief, 5\u00a0g of hydrolyzed sample was mixed with 25\u00a0mL of acid water. The extracts were filtered and passed through an SCX cartridge, previously conditioned according to manufacturer protocol . The obtained solution was dried under N2. Each dried residue was dissolved in 60 \u00b5L water and 40\u00a0\u00b5L ethanol/pyridine (4:1). Five \u00b5L ethyl chloroformate was added to the mixture. Finally, 150\u00a0\u00b5L of chloroform was added.Derivatized samples were analyzed using the GC system  equipped with an FID. Separation of compounds was conducted on a 10\u00a0m CP-Sil 19 capillary column  using nitrogen 5.0 as the carrier gas . The injection volume was 1 \u00b5L, and the flow rate was set at 0.7\u00a0mL/min. The injector temperature was 250\u00a0\u00b0C with a split ratio of 50:1, and the FID temperature was 250\u00a0\u00b0C. The oven temperature was programmed as follows: the column was held initially at 140\u00a0\u00b0C, then increased to 280\u00a0\u00b0C at 40\u00a0\u00b0C/min and held for 3\u00a0min. Chromatographic data were recorded and integrated using Clarity software  [Blood samples were collected from all experimental animals to determine haematological  and biochemical parameters . Spectrometric analyses were performed by a Konelab T20xt biochemical analyzer (Thermo Fisher Scientific) and commercially available reagents, according to . FurtherColorimetric assay of determination of MDA and GSH / GSSG was carried out according to the manufacturer protocol .From each slaughtered pig, the entire liver and kidneys were removed and weighed, then the mass of these organs was converted to the percentage of the live animal weight. Tissue samples of the liver  and the intestine  were collected and immediately fixed in 10% formaldehyde solution to investigate and evaluate pathomorphological changes. Fragments of tissues were cut at 3.0\u00a0\u03bcm, then positioned onto Superfrost Plus slides  with the orientation core placed up on the slide. All tissue blocks were oriented the same way; then, the entire tissue block was cut with the remaining sections dipped in wax and stored at room temperature. The sections were stained with hematoxylin and eosin according to standard procedures. Pictures were taken using an inverted Olympus microscope IX 71S8F-3  at the magnification 10\u201320\u2009\u00d7\u2009for liver samples and 10\u2009\u00d7\u2009magnification for jejunum.p\u2009<\u20090.05 were considered significant.The data were analyzed using STATISTICA.CZ, version 12.0 (Czech Republic). The results were expressed as a mean from all samples\u2009\u00b1\u2009standard deviation (n\u2009=\u20093). Statistical significance was determined by examining the basic differences among groups using ANOVA and Schaffer's method for all parameters. The differences with"}
+{"text": "A probabilistic model for random hypergraphs is introduced to represent unary, binary and higher order interactions among objects in real-world problems. This model is an extension of the latent class analysis model that introduces two clustering structures for hyperedges and captures variation in the size of hyperedges. An expectation maximization algorithm with minorization maximization steps is developed to perform parameter estimation. Model selection using Bayesian Information Criterion is proposed. The model is applied to simulated data and two real-world data sets where interesting results are obtained. A large number of random graph models have been proposed . Two-mode networks consist of two different kinds of vertices and edges can only be observed between the two types of vertices, but not between vertices of the same type. A hypergraph can be represented as a two-mode network by considering the hyperedges as a second type of vertices. For example, an equivalent bipartite representation of the hypergraph shown in Fig.\u00a0Two-mode networks have been studied in various disciplines including computer science  model for random hypergraphs, which is a natural extension of the Latent Class Analysis (LCA) model  model  model which builds on the proportionality assumption on the conditional probabilities.While the LCA model captures the clustering and heterogeneity of hyperedges in real world data sets, a large number of latent classes are typically required to achieve a good fit of the data. As a result, the number of parameters grows quickly with a moderate or large number of nodes. The complexity of the LCA model can be substantially reduced if we assume that some of the latent class conditional probabilities K dimensional vector, the ELCA model assumes that the latent class conditional probabilities are of the form Let a priori independent. Thus, the probability that a hyperedge has primary cluster label g and additional cluster label k is g,\u00a0k) is g is Let G primary clusters and K additional clusters, the probability of observing a hyperedge k, then the likelihood function is invariant under the transformation C is some positive constant such that Under the ELCA model with k and G primary clusters and K additional clusters can be equivalently represented as a standard LCA with G sets of equal size K, and We define the G primary clusters and K additional clusters, the number of parameters is given by It is easy to see that under the proportionality assumption, the ELCA model achieves significant reduction in the number of parameters. For the ELCA model with We analyze the distribution of the size of a random hyperedge under the proposed ELCA model. Proposition\u00a0N vertices. Suppose the condition Suppose we are given the LCA model with parameters A denote the cardinality B denote the cardinality Let The proof is straightforward and is given in the Appendix. G cluster LCA model. Similarly, we let G clusters and K additional clusters. The following result can be derived.We now let G components.Under the specifications of a LCA model with parameters Under the specification of a ELCA model with parameters g, , and an overall complexity of For the E-step, we need to evaluate the expected complete data log-likelihood, which is the expectation of  conditioa do not have closed form solutions. We utilize the MM algorithm  Schwarz  to deterWe conduct simulation studies to examine the performance of the proposed EM algorithm for the ELCA model and the behavior of BIC as a model selection criterion. The results presented in Tables\u00a0a, For the model parameters mentclass2pt{minimM increases.We examine the performance of BIC in choosing the optimal number of primary and additional clusters. The values in Tables\u00a0N increases, the proportion of times that the true model is recovered increases considerably. On the other hand, there is no clear relationship between the number of hyperedges M and the proportion of successful recovery of the true model.As a final simulation study, we simulate hyperedges from the LCA models with two and three clusters and note that they are special cases of the ELCA models with Our first application is modeling co-appearance of the main characters in the scenes of the movie \u201cStar Wars: A New Hope\u201d. We collected the scripts of the movie from the Internet Movie Script DatabaseWe determine the optimal number of clusters and additional clusters using BIC where the results are provided in Table\u00a0The results from fitting the ELCA model with The estimates The estimated primary cluster assignment probabilities from the EM algorithm for each movie scene in the Star Wars movie are shown in chronological order in Fig.\u00a0The uncertainties in primary clustering are also illustrated in a ternary plot in Fig.\u00a0The estimated additional cluster assignment probabilities for each movie scene in the Star Wars movie are shown in chronological order in Fig.\u00a0As a comparison, the results from fitting the standard LCA model with 3 clusters are shown in Tables\u00a0The difference in the clustering structure between the ELCA model and the LCA model is expected as the ELCA model explicitly captures the variation in the size of hyperedges. In comparison, the LCA model cannot decouple the variation in the size of hyperedges from the primary clustering structure. This is a key advantage of the ELCA model where the underlying structure of the size of the hyperedges can be uncovered. Furthermore, as a constrained version of the LCA model with 6 clusters, the ELCA model with 3 primary clusters and 2 additional clusters is far more parsimonious.As a second application of the ELCA model, we collected news articles published by ReutersThe model with 5 clusters and 2 additional clusters was chosen by the BIC and fitted to the data set. The BIC scores for a range of models are shown in Table\u00a0The parameter estimates The clustering structure can be deduced from the estimate We have proposed the Extended Latent Class Analysis model as a generative model for random hypergraphs. Building on a proportionality assumption, the ELCA model introduces two clustering structures for hyperedges which captures variation in the size of hyperedges. The model achieves significant reduction in model complexity compared to the standard Latent Class Analysis model. An EM algorithm has been developed for model fitting where the M-step involves a series of conditional maximization and model selection is performed using BIC. The proposed model is fitted to two data sets and this yields interesting and interpretable structure within the vertices and hyperedges.Several extensions to the ELCA model are possible. Hyperedges typically have temporal information associated with them, which is the case for the two applications in this paper. Developing a hypergraph model to incorporate such temporal information is of interest. Furthermore, while the ELCA is developed in the context of hypergraph applications, the model could be useful in other applications where the proportionality assumption on latent class conditional probabilities is plausible."}
+{"text": "Time has always been a source of perplexity and fascination for human beings. Presocratic philosophers initiated the first discussions on the reality of time and its relation to change. Heraclitus presumed that change was a basic and irreducible ingredient of nature. According to him, the world would be a manifold of substances in permanent change. Parmenides, on the contrary, famously denied change. He argued, in what was possibly the first deductive argument in the history of ontology, that change is impossible because it demands that what is not, should somehow be. He relentlessly concluded that our image of a dynamic universe is a pure illusion: reality is fixed, coming to be and perishing are excluded from the cosmos, and whatever exists must be permanent  exist. They form a four-dimensional \u201cblock\u201d of spacetime. Events are ordered by relations of earlier than, later than, or simultaneous with, one another. These relations among events are unchanging. They cannot change because time Dean Buonomano has recently pointed out in a discussion with Carlo Rovelli that:\u201c(\u2026) unlike the empirically confirmed predictions of relativity , it is important to stress that there is no empirical evidence for the block universe. Indeed, it is far from clear that there are any testable predictions that could prove or disprove the existence of the block universe (other than the emergence of a confirmed time traveler).\u201d . Similarly, they do not perceive space but spatial relations among things. In particular, we do not perceive the passage of time. We perceive how our brain changes. I claim that there is no present  Romero, .Perhaps this can be expressed more simply by saying that what we call consciousness arises from groups of successive brain events arranged in some specific configurations in spacetime. Those events are changes and processes, that is, chains of pairs of states that associate properties of one part of the brain with properties of other regions, either in the same brain or in the local environment, at slightly different times. Since these properties are not the same across the time dimension, the illusion of a \u201ctime flow\u201d arises. However, time does not flow in any meaningful, non-metaphorical sense. It is just one dimension along which spatial properties vary. The \u201cflow\u201d of time is just a brain construct, an illusion, albeit a very stubborn one because it is rooted in what defines our very identity. The variation, I insist, is only a relative difference in the distribution of properties along the manifold that represents the four-dimensional spacetime.Physical time within human time,\u201d Gruber et al. . This is achieved with the immersion of the subject in a virtual reality fed with a system of cameras whose output is controlled and allows the researcher to switch between present, past, or future moments. The resulting \u201cpresent\u201d experienced is not unique and hence not a property of spacetime but rather of the specific IGUS. This is a very important result obtained by Gruber et al.: the two diverging ideas of time, the physical, objective time and the human, subjective one, are the result of the same and unique set of physical laws. The neuroscience and physics of time seem to accord through mechanisms that can be objectively tested.Further experiments should evaluate the efficacy of different IGUS configurations to implement tasks related to the survival skills of the individual. Complexity and a variety of new tests can be obtained by introducing various gadgets. The comparison of the results of such research might lend support to the hypothesis advanced by Hartle  that theare.\u201d  the present does not flow or move. Only material individuals  can change and move. Becoming is not a property of physical events but of the consciousness of the events. We call \u2018becoming' to the series of states of consciousness associated with a certain string of physical changes. Events do not become. Events just  Romero, .The author confirms being the sole contributor of this work and has approved it for publication."}
+{"text": "Their low incidence, and the complexity of their clinico-pathological characteristics demand standardized, cancer-tailored diagnostics and therapies managed at high-volume, multidisciplinary care centers. This study evaluates the quality of STS management in north-east Italy (Veneto Region) through a list of This population-based study concerns all incident cases of STS in 2018 (214 cases) recorded in the adult population censored by the Veneto\u2019s regional Cancer Registry. Based on the international literature, a multidisciplinary working group of experts identified a set of indicators for monitoring the quality of diagnostic, therapeutic, and end-of-life clinical interventions. The quality of care was assessed by comparing the reference thresholds with the indicators\u2019 values achieved in clinical practice.Diagnostic procedures showed poor adherence to the thresholds, with a low percentage of histological diagnoses validated by a second opinion. The indicators relating to the surgical treatment of superficial, small, low-grade STS, or of medium, high-grade STS of the head\u2013neck, trunk, or limbs were consistent with the thresholds, while for intermediate, high-grade  and retroperitoneal STS they fell significantly below the thresholds.A critical evaluation of the clinical indicators allowed to uncover the procedures needing corrective action. Monitoring clinical care indicators improves cancer care, confirms the importance of managing rare cancers at highly specialized, high-volume centers, and promotes the ethical sustainability of the healthcare system. Soft tissue sarcomas (STS) account for less than 2% of all malignant tumors in adults. They include a wide range of different malignancies , classifIn cancer care, adopting standardized diagnostic and therapeutic strategies enables care quality to be monitored consistently, and healthcare system efficiency to be critically addressed. Both these actions are essential to improving patient outcomes and optimizing the allocation of resources . VariabiQuality of patient care can be reliably monitored by means of \u201cindicators\u201d addressing a center\u2019s performance, and the outcomes of the diagnostic-therapeutic pathway adopted. In oncology, as in other clinical specialties, quality indicators are useful to both identify and quantify: (i) the appropriateness of diagnostic procedures; (ii) the efficacy of anticancer therapies and surgical treatments; (iii) the critical areas most requiring corrective actions; and (iv) the sustainability and relative priority of investments directed toward oncological cares , 10.This population-based study critically addresses the clinical management of STS patients\u2019 resident in the Veneto region of Italy in the year 2018. Quality of care was ranked thon the strength of a set of internationally-acknowledged clinical indicators selected by a multidisciplinary regional working group (RWG) of specialists with expertise in soft tissue malignancies.The Italian National Health System is a public service supported primarily by general taxation and it is managed on a regional basis. Its policies are grounded on the fundamental values of universality, free access, freedom of choice, pluralism in provision, and equity.In 2015, the Veneto\u2019s Regional Oncology Network, comprising Veneto Oncology Institute, a center included in the European network for Rare adult solid Cancer (EURACAN), produced a comprehensive document detailing the clinical procedures to be applied in each step of the clinical management of STS patients, from their initial diagnosis to their end-of-life care . The VenData were collected from the population-based Veneto Cancer Registry, a high-resolution database which covers the population of the whole region (4.9 million residents), and from the regional health service records. The present study concerns all incident cases of STS recorded by the Registry in the year 2018. Recording procedures rely on various informative sources, such as pathology reports, clinical charts, death certificates, and health service administrative records. The variables available for STS include: age and sex; tumor site ; diameter of the primary tumor (mm); depth ; histological subtype (ICD-O-3 code); tumor grade ; combined clinical-pathological TNM stage at diagnosis ; treatments ; and status of resection margins . The clinical information recorded also includes: the results of diagnostic imaging ; the identification code of the institution(s) delivering the treatment; and the timing of therapeutic procedures .In 2021, a RWG of epidemiologists, healthcare managers, oncologists, pathologists, radiologists, radiotherapists, statisticians, and surgeons established a list of indicators to use in monitoring the quality of care provided for adult STS patients, the consistency between threshold values adopted for these indicators, and how centers performed in real-world clinical practice. All nine regional public health institutions potentially involved in STS care were included in this quality assessment (QA) project.Based on the different steps of a patient\u2019s clinical management , 22, theIn 2018, the regional population-based cancer registry (censoring the whole Veneto population of nearly 5 million) recorded 214 incident cases of adult STS. Pre-biopsy imaging, as recommended by the international and regional guidelines, was available for 89% of patients. The initial diagnosis of STS was supported by a second opinion in 45% of cases. Both these indicators fell below the established thresholds.These two performance indicators measure whether and when the activities recommended in order to accomplish the strategic objectives of the integrated care processes actually took place . More thTwo of the four indicators relating to surgical therapies  were consistent with the thresholds. Among the 2018 incident cases of retroperitoneal STS, however, only 37% were treated with multivisceral surgery .None of the indicators relating to combined therapies , or medical therapies  were consistent with the thresholds. The best performance (75 vs. a threshold of 80%) was achieved for indicator No. 11 relating to how multimodal therapies were administered for head\u2013neck, trunk, or limb STS of any size or histological grade. The prevalence of ChT withdrawal due to toxicity was 2%.More than 30% of patients were given ChT within 30\u2009days before their death.ad hoc defined indicators concerning all the main clinical phases of STS care: diagnosis, process performance, surgical/medical treatments, and end-of-life care. The benchmark value for of the indicators established by an interdisciplinary regional group of experts was compared with the value obtained in real-world clinical practice.This study assessed how the Veneto healthcare system performed in the clinical management of 214 incident cases of adult STS diagnosed in 2018. The evaluation was conducted with a list of A suboptimal management of the diagnostic procedures came to light as concerned both the proportion of STS cases assessed on imaging before a biopsy was performed , and the proportion of histological diagnoses confirmed by a second opinion .The gross features of the tumor  and the microscopic phenotype are determinants in STS care. Diagnostic procedures chiefly involve two methods: imaging ; and pathology . As a rule, however, the conclusive assessment demands a critical merging of both. The high proportion of STS patients who underwent imaging only after a biopsy  is a concern because the lack of information from propaedeutic imaging data significantly limits the pathologist\u2019s assessment. Mathoulin-P\u00e9lissier et al. , 25 mentThe low incidence of STS, and the variety of histological subtypes, significantly limit operators\u2019 experience with its diagnosis. This results in high rates of intra/inter-observer variability . CompariThe RWG\u2019s process performance indicators measured the quality of the integrated healthcare services for STS, revealing a partial misalignment between the expected threshold values and the evidence on actual clinical patient management in 2018. The proportion of patients treated within 3\u2009months of receiving their histological diagnosis was compliant with the 90% expected threshold. However, a high proportion of patients were not treated entirely at reference centers. The timely referral of patients with rare cancers to specialized institutions for all their care reduces the time elapsing between diagnosis and therapy, increasing the efficacy and efficiency of treatments.The results emerging from this study point to the need for a combined strategy involving healthcare managers, physicians, and patients (or patients\u2019 associations), to ensure that: (i) public healthcare policy promotes dedicated care strategies for low-incident cancers; (ii) healthcare institutions disseminate consistent information to promote patients\u2019 awareness. The patient-physician therapeutic alliance demands transparency in the delivery of information  on the dFour of the indicators concerned surgical treatments , three concerned medical treatments , and three  addressed multimodal  therapies.Of the first four indicators , two were entirely consistent with the thresholds. In other words: superficial, small-sized or low-grade STS received appropriate surgical treatment ; surgery for medium- or high-grade STS of the head\u2013neck, trunk or limbs was radical only in 80% of cases (indicator No.7). The fourth, concerning surgery for retroperitoneal STS, was the indicator with the most significant gap between the threshold (>80%). The proportion of patients who actually had multivisceral surgery is only 37%. This result may be  explained by three main considerations. One concerns the fact that multivisceral surgery  has beenThe indicators referring to the treatment of large-size STS (Nos. 9 and 10) showed a suboptimal care management . These significant gaps between the expectations and the results obtained demands a critical reassessment of either the diagnostic-therapeutic procedures implemented or the consistency of the indicators\u2019 thresholds. This need is reinforced by the unsatisfactory values obtained for indicators Nos. 12 and 13, both of which address the timely treatment of large-sized STS after surgery. These data could also be explained by the still high proportion of patients treated not at a referral center in our Region.In the terminal phase of their disease (in the 30\u2009days before they died), 31% of the patients considered were given chemotherapy (threshold <10%). This finding prompts both clinical and ethical considerations. The clinical issue concerns prioritizing corrective action to ensure the cost-effectiveness of anticancer drugs, balancing the therapeutic stress and the effectiveness of the clinical advantages. The ethical issue mainly concerns clinicians\u2019 empathy when dealing with patients\u2019 wishes in the most advanced phase of their illness .This study has its strengths and weaknesses. The main weakness lies in the small number of cases considered, resulting in a limited number of STS in each diagnostic category. Given the heterogeneous clinicopathological outcome of STS subtypes, this situation can potentially result in significant biases. More extensive studies should address performance in care provision for early vs. more advanced STS, which could be affected by the biological profile of the malignancies considered, inconsistent clinical management, or (even by) a suboptimal choice of performance indicators. The currently available indicators do not include any quality assessment involving molecular testing, or any reference to personalized therapies, though their impact is already crucial to high-standard patient management .The study\u2019s main strength lies in its population-based (not center-specific) design, so it potentially represents the quality of care offered to a large, epidemiologically stable population. Identifying which indicators did or did not reach the  thresholds established by our RWG represents a good starting point for critically addressing what has been achieved so far, and what we need to be done as we move forward.In conclusion, this study emphasizes that the evaluation of care pathways is critical to ensure quality improvement and excellence of care at all times, especially for rare cancers such as soft tissue sarcomas. In this way, procedures in need of corrective action can be identified and new evidence-based goals can be formulated to improve patient management, outcomes, and optimize resource allocation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Veneto Oncological Institute. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MRu and ABu: conceptualization and review and editing. ABru, MS, ST, GG, CC, MZ, and PF: data curation. ABu, CC, and ABra: formal analysis. CR and SM: funding acquisition. ABu: investigation. ABu and VB: methodology and project administration. ABu, ABru, CR, and SM: resources. CC: software. MRu, AD, ABo, PB, LD, MRa, CR, and SM: supervision. ABu, MZ, AV, AS, and ABru: writing original draft. All authors contributed to the article and approved the submitted version.This research has received \u201cCurrent Research\u201d funds from the Italian Ministry of Health to cover publication costs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The authors regret that the email address of the corresponding author (gzsongm@126.com) was not shown in the original manuscript. The corrected list of affiliations is as shown here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."}
+{"text": "Correction to: Clinical Epigenetics (2023) 15:126 10.1186/s13148-023-01536-3Following publication of the original article , the autThis has now been corrected with this erratum.The original article has been corrected."}
+{"text": "Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These conditions may involve genes in difficult-to-align genomic regions that are refractory to short read approaches. Structural variants in these regions can be particularly hard to detect or define with short reads, yet may account for a significant number of cases. Long read sequencing can overcome these difficulties but so far little data is available regarding the specific analytical challenges inherent in such regions, which need to be taken into account to ensure that variants are correctly identified. Research into chronic fatigue disorders faces the additional challenge that the heterogeneous patient populations likely encompass multiple aetiologies with overlapping symptoms, rather than a single disease entity, such that each individual abnormality may lack statistical significance within a larger sample. Better delineation of patient subgroups is needed to target research and treatment.AKR1C gene locus, which was indicated but could not be resolved by short-read sequencing. We then use GC\u2013MS/MS serum steroid analysis to investigate the functional consequences.We use nanopore sequencing in a case of unexplained severe fatigue to identify and fully characterise a large inversion in a highly homologous region spanning the Several commonly used bioinformatics tools are confounded by the homology but a combined approach including visual inspection allows the variant to be accurately resolved. The DNA inversion appears to increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory GABAergic neurosteroids and impaired progesterone metabolism which could suppress neuronal activity and interfere with cellular function in a wide range of tissues.This study provides an example of how long read sequencing can improve diagnostic yield in research and clinical care, and highlights some of the analytical challenges presented by regions containing tandem arrays of genes. It also proposes a novel gene associated with a novel disease aetiology that may be an underlying cause of complex chronic fatigue. It reveals biomarkers that could now be assessed in a larger cohort, potentially identifying a subset of patients who might respond to treatments suggested by the aetiology.The online version contains supplementary material available at 10.1186/s12967-023-04711-5. The dramatic reduction in the cost of high-throughput sequencing over recent years has made short-read whole genome sequencing (WGS) viable as a go-to option for clinical genomic analysis. The high accuracy at base level combined with increasingly sophisticated bioinformatic analytical tools has enabled diagnosis in many cases that were previously unexplained. Despite these advances, however, causative variants cannot be found for up to three quarters of patients thought to have a genetic disorder , leavingDisorders of unexplained chronic fatigue (CF), such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), show strong evidence of heritability with multiple cases in single families, but the inheritance pattern does not appear to be Mendelian , 3. So fThere is hope that the rise of next-generation WGS and large-scale genome-wide association studies (GWAS) will bring about a step change in understanding. However, financial constraints mean that GWAS must generally rely on a single nucleotide variant (SNV) array based approach, which by definition cannot identify structural variants (SVs), novel SNVs, or any other SNVs not selected for the array panel. Next-generation WGS may also fall short because some parts of the genome are inherently \u201cdark\u201d to short-read alignment due to high homology or low complexity, where entire reads can map equally well to multiple loci. These regions may be more prone to structural variation and are thought to account for a significant proportion of pathogenic variants , 6.AKR1C1-4 lie in numerical order, with AKR1C1 on the forward strand facing AKR1C2 on the reverse strand \u00a0then\u00a0AKR1C3 and AKR1C4 both on the forward strand. The four genes share\u2009>\u200986% amino acid sequence identity overall, while AKR1C1 and AKR1C2 in particular share nearly 98% amino acid sequence identity and differ by only 7 amino acids [AR) in a similar way to benzodiazepine sedatives [AKR1C1 and 1C2 have been surprisingly little studied in any disease context at all [One such region is a stretch of segmental duplication on human chromosome 10p15 to 10p14 that contains the four Aldo\u2013Keto Reductase Family 1 Member C genes. t at all .Long read sequencing (LRS) technologies such as those from Oxford Nanopore (ONT) and Pacific Biosciences (PacBio) have the potential to shed light on these dark regions. Long reads are typically in excess of 10 kb in length and can reach several megabases , meaningUntil recently, the relatively low base-level accuracy and high cost of LRS have not permitted clinical adoption, but advances in sequencing chemistry and base calling have now improved accuracy to acceptable levels for the majority of applications , 12 and Here, we demonstrate the power of LRS to fully characterise a large inversion in a difficult-to-align region that is all but invisible to short reads. We highlight some of the challenges that can arise when analysing both long read and short read data from such regions, and show that even when SV callers do indicate structural variation they may not correctly resolve the SV, potentially suggesting a functional impact that can differ significantly from the true effect.AR, resulting in a complex, multi-system syndrome of severe fatigue. We further propose that the genetic and biochemical biomarkers discovered in our patient could be assessed in a larger cohort, to see if they constitute a subset of CF patients who might respond to treatments suggested by the aetiology.We also present biochemical evidence that the variant described here is functional in our patient. We propose that it represents a novel aetiology combining underactive AKR1C1 with overactive AKR1C2, which limits the clearance of intracellular progesterone while suppressing neuronal activity via the GABAThe 42 year old patient has a long and unusually complex history, of which the most debilitating aspect is increasingly severe mental and physical fatigue. Onset occurred after a viral infection age 16, initially mild but gradually progressing. At present, the patient has been completely housebound for many years and is unable to sit up or speak for much of the day.Clinical diagnoses include central adrenal insufficiency and partial central diabetes insipidus, both thought to be hypothalamic rather than pituitary, along with renal salt wasting and functional pan-enteric dysmotility. There was previously a diagnosis of ME/CFS for some years but that label can be a barrier to getting appropriate treatment and was removed after the later diagnosis of other conditions known to cause persistent fatigue. Mental health assessment concluded that there was no underlying psychiatric disease. None of the current diagnoses, however, fully explains the severity of illness or the full range of symptoms. It is also highly unusual to see this constellation of conditions, especially with young-adult onset. The patient was referred to the UK\u2019s 100,000 Genomes Project (100kGP) on suspicion of Gitelman syndrome due to the clinical presentation of renal salt wasting, but no likely tubulopathy-associated variants have been found and other aspects of her condition are outside the scope of the 100kGP.AR, possibly due to upregulation of the metabolic pathways that produce inhibitory GABAergic neurosteroids, which could be either genetic or acquired. The patient reported that a similar but less acute sense of sedation, distinct from the fatigue, had started some years previously. It had originally occurred only in the latter stages of the menstrual cycle but had progressively spread throughout the cycle until it is now constantly present, although still with some cyclic variation.Of particular note was that certain steroid medications provoked an atypical reaction resembling the effects of benzodiazepines. This inspired a novel hypothesis of excessive neurosteroid-mediated activation of the GABAThe family history was suggestive of a genetic predisposition to CF, with unexplained fatigue in several family members , including a period of quite debilitating fatigue in the mother\u2019s early 20s and a maternal second cousin diagnosed with ME/CFS.AKR1Cs in relation to a different condition that the patient also has in a mild form, which she joined after the discovery of the inversion in the 100kGP data. The results from a luteal phase sample showed substantial overexpression of AKR1C2 mRNA in stabilised whole blood, with relative expression approximately 100 times higher than controls, while AKR1C1 and AKR1C3 were comparable with controls (full details seen but not reproduced here as the other study has not yet reported).RQ-PCR was conducted for a separate study investigating the Whole genome sequence data for the patient and the patient\u2019s mother were provided by the 100kGP. The data were produced via the standard 100kGP pipeline  and CanvDNA was extracted from 1 mL of whole blood with the Nanobind CBB Big DNA kit (Circulomics NB-900-001-01). After extraction, the genomic DNA (gDNA) was homogenised by 3\u201310 passes of needle shearing (26G) and 1h of incubation at 50\u00a0\u00b0C. The gDNA was then split into two aliquots. 4 \u03bcg of gDNA were fragmented to\u2009~50kb with the Megaruptor 3  and 10 \u03bcg were fragmented to 20-25kb with a gTUBE . All samples were depleted of short DNA fragments (less than 10kb long) with the SRE kit . DNA size distributions were assessed at each step on a FemtoPulse system  and all samples were quantified on a Qubit fluorometer .Two sequencing libraries were generated from 1.5 \u03bcg of each gDNA sample with the SQK-LSK109 kit (Oxford Nanopore Technologies). 25 fmol of the obtained sequencing libraries were loaded onto a R9.4 flow cell (ONT) and sequenced on a PromethION48 (ONT). Both flowcells generated approximately 90Gb of mappable data, providing a total sequencing depth of\u2009~60x. The N50s were 23kb and 37kb.Base-calling was performed with Guppy-4.0.11 using the high accuracy model and a qscore filter of 7. Full details of the protocol are given in Additional file The variant call files provided by the 100kGP were annotated and filtered using seqr version 0.3.0 .Identifying and defining the inversionPhasingAssembling the allelesAnnotating the allelesComparing the long read data to the short readsAnalysis of the ONT data followed the workflow:Visual inspection in Integrative Genomics Viewer (IGV)  was usedThe patient provided five samples in total: two from the early follicular phase of the menstrual cycle and three from the mid luteal phase, covering different cycles with symptomatic variation. All samples were taken in the morning. Blood was drawn from the cubital vein, centrifuged at 1790g for 10 min, and the serum stored at \u221220\u00a0\u00b0C until analysis.The samples were prepared and analysed using our previously described GC\u2013MS/MS method . In brie82 analytes were reported and concentrations were compared against a pool of controls, who were selected from our records to correspond as closely as possible to the patient. These were healthy, medication-free, premenopausal women, matched to the relevant stage of the menstrual cycle . The control data were assessed as medians with interquartile range to allow for skewed distributions and non-constant variance.AKR1C1 to part way through the extended 5\u2019UTR of the first two transcripts of AKR1C2 , in the second intron of the first transcript of Both variants are also present in the patient\u2019s mother, suggesting that they are on the same allele. The splice-site SNV was therefore deemed unlikely to be relevant, since the inversion effectively removes the first exon of the extended 5\u2019UTR meaning that transcript 1 would not be produced. This SNV is also relatively common, with an ALFA Allele Frequency of 0.12712 in Europeans (lower in other populations). It is not listed in ClinVar.Detailed analysis of the inversion was not possible with the short-read data due to the high homology. In addition, similar large inversion calls were present in a number of unrelated individuals in the 100kGP dataset, so that it was unclear whether the inversion was actually real or merely an alignment artefact caused by the homology. Long read sequencing was therefore sought instead.AKR1C1 and AKR1C2 but does not detect an inversion. On visual inspection in IGV, however, signs of a heterozygous large SV spanning the region can clearly be seen, although the exact nature of the SV is not immediately obvious  of between 50 and 420 bp in the region of After initial phasing with Samtools Phase it can be seen that the REF allele looks normal while the ALT allele has several features suggesting an inversion, including mirrored insertions and deletions where a segment is deleted from one side of the region and the reverse complement of that segment inserted at the equivalent position on the other side. These are the indels that Sniffles had detected. Due to the high homology, however, there is not the classic inversion pattern of reads split into supplementary alignments on opposite strands. Instead, the reads remain intact but with clusters of high-density SNVs interspersed with sections that appear normal, as well as the mirrored indels.AKR1C1 and 602 bp upstream of the first coding exon of AKR1C2.Breakpoints were estimated by the loci at which variants in the ONT sequence started to differ significantly from those in the 100kGP sequence. To confirm the inversion we created a new reference sequence consisting of the GRCh38 consensus sequence with the region between the proposed breakpoints inverted. When mapped to this inverted reference the ALT allele fits cleanly while the REF allele now shows the same pattern of variants as the ALT allele had when aligned to the standard reference, but with the pattern reversed , but in a slightly different position because repetitive sequence at the end affects short-read alignment.Attempts to assemble the ALT allele using Canu and minimap2-miniasm appeared to be confounded by the homology in the region but good results were obtained using Flye, either with 5 rounds of polishing as part of the Flye pipeline or with one round of polishing by Flye followed by one round of polishing with racon. Accuracy was assessed by mapping the raw reads back to the assembly for visual inspection in IGV  to each of the assembled alleles. As can be seen in Fig.\u00a0The inversion revealed by the ONT sequence is nearly 10 kb shorter than suggested by the short read data. To investigate this discrepancy we created two simulated reads from the 10 kb sections immediately outside of the breakpoints at each end of the ALT assembly. These simulated reads were then aligned back to the GRCh38 reference both in their correct positions and at the opposite sides Fig.\u00a0a. This eAKR1C1 side appears to have been copied and pasted over the equivalent position on the AKR1C2 side  activity : transcripts 1 & 2 (CCDS7062) differ only in their 5\u2019UTR and both encode the full AKR1C2 protein of 323 amino acids (isoform 1). Transcript 3 (CCDS44350) encodes isoform 2, a truncated version of only 139 amino acids that is predicted to be inactive\u00a0AKR1C1 has only one transcript in the CCDS. & 2 CCDS062 diffeAKR1C1 with the coding region of AKR1C2. This has a number of consequences:AKR1C2 transcripts 1 & 2 are still in their correct place, meaning that transcription could presumably be initiated in the normal way and so potentially result in a rearranged AKR1C1 mRNA with those additional exons and possible retained intron added at the 5\u2019 end. It seems likely that this would affect expression and/or translation of AKR1C1.The initial non-coding exons of AKR1C2 would only have transcript variants 3, 4 and 5. Transcripts 4 and 5 would presumably still produce a functional protein but it seems likely that expression would be altered.Without those first non-coding exons, AKR1C1 and 1C2 are reversed relative to AKR1C6P, AKR1C3, AKR1C8P, and to all of the regulatory regions outside of the breakpoints. These include three transcriptional cis-regulatory regions on the forward strand in the RefSeq curated subset, marked A, B and C in Fig.\u00a0AKR1C1 with high confidence in K562 cells. The middle of these (B) also positively regulates AKR1C2. GeneHancer indicates other regulatory elements with interactions potentially disrupted by the inversion, as does the ORegAnno database  from 5\u03b1-DHP, 5\u03b2-DHP and 5\u03b1-dihydrodeoxycorticosterone (DHDOC) respectively , 28. Allhe brain , 29, 30.AR [AKR1C1 acts predominantly as a 20\u03b1-reductase (20\u03b1-R) that reduces active steroids to their inactive metabolites, particularly P to 20\u03b1-DHP. AKR1C1 is also thought to be the main enzyme producing the 3\u03b2 metabolites of 5\u03b1-reduced steroids ; these iAR , 30.If the inversion suppresses AKR1C1 while upregulating AKR1C2 then these properties could fit well with the clinical presentation. As shown in Fig.\u00a0The overlapping activities of the AKR1Cs and the complex pathways of steroid metabolism more widely can make it challenging to assess individual enzymes from peripheral blood. Hepatic AKR1C4 in particular has high catalytic efficiency for both 20\u03b1- and 3\u03b1- reduction  and woulThe high ratio of /iso directly supports the hypothesis of a relative increase in inhibitory GABAergic neurosteroids in our patient, with the higher value in the luteal phase corresponding to increased symptoms in the latter part of the menstrual cycle.The high ratios of 17-OHP/17,20\u03b1-diOHP and follicular P/20\u03b1-DHP are consistent with partial loss of AKR1C1. The normal-low luteal P/20\u03b1-DHP ratio is surprising but could be explained by compensatory diversion of P down the 5\u03b1 pathway.5\u03b1-R is ubiquitous while the majority of 5\u03b2-reduction occurs in the liver , meaningAKR1C2 mRNA in luteal phase also indicates that AKR1C2 activity may be increased.The low ratio of DHT/Adiol is consistent with increased 3\u03b1-reduction by AKR1C2, with the caveat that adiol can also be synthesised from allo, via androsterone, bypassing DHT and potentially altering the DHT/Adiol ratio. The overexpressed AR suggested by the clinical presentation, and indicate that this is due to overactive AKR1C2 combined with partial loss of AKR1C1. It seems possible that for AKR1C2 the inversion either creates an enhancer or removes a silencer. RQ-PCR did not find low AKR1C1 mRNA expression, as might be expected with the underactive enzyme, but expression could be tissue-specific. It is also possible that gene expression is normal but the extra 5\u2019UTR exons, with possible retained intron, interfere with translation and so cause loss of protein expression or function on that allele.All together, these results support the hypothesis of excessive neurosteroid-mediated activation of the GABAAR are all active in the gastrointestinal tract, where GABA is involved in regulating secretion and motility [If this variant is indeed causing overactivity of AKR1C2 while limiting AKR1C1 then it could be part of the underlying cause not only of the fatigue and sedation, due to the sedative effects of allo and preg, but also of the endocrine, renal and gastrointestinal dysfunction in this patient. Excessive GABAergic inhibition in the hypothalamus could suppress the release of corticotropin releasing hormone (CRH) and antidiuretic hormone (ADH), resulting in cortisol deficiency with diabetes insipidus , 36. AKRmotility , 38, 41.AKR1C1 and 1C2 could potentially affect any tissue where steroid hormones undergo 20\u03b1- or 3,5-reduction, with implications for a variety of conditions where the aetiology is unknown or a subset of cases remains unexplained. This would be particularly the case for multi-system chronic fatigue disorders like ME/CFS, fibromyalgia and long covid. Gastrointestinal dysfunction and hypocortisolism are common in these conditions, as is dysregulation of the autonomic nervous system [More broadly, variants in s system \u201344 whichs system , 46. Thes system , 48, as s system .AKR1C1 mRNA has the extra 5\u2019UTR exons and retained intron, and to assess the relative expression of the two genes and their isoforms. In vitro functional studies would permit direct investigation of the effect of the inversion on protein expression and function, and further metabolomic analysis, if possible, could assess analytes that would give a more direct indication of AKR1C2 activity in neurosteroid synthesis but\u00a0which could not be accurately measured with GC\u2013MS/MS, such as 5\u03b1-DHP, 5\u03b2-DHP, DHDOC and THDOC. We also hope to extend the study to other patients, ideally in collaboration with large-scale projects such as DecodeME [AKR1C1 & 1C2 and consequent alterations in steroid metabolism are associated with disease in a subset of patients.We therefore plan further investigations, including RNA-seq to ascertain whether or not the variant DecodeME , to inveAR modulating steroid antagonist GR3027 (Golexanolone) are showing promise in clinical trials for a variety of conditions associated with relative allo excess [If this hypothesis is correct then it also raises the possibility of treatment. The commonly used and well tolerated bile acid ursodeoxycholic acid (UDCA) is a selective AKR1C2 inhibitor that can penetrate the brain , 51, ando excess .This study has some limitations. As a single case without functional studies it cannot demonstrate conclusively that the inversion is the direct cause of the biochemical abnormalities, or that those abnormalities do indeed underlie the patient\u2019s symptoms. The relatively small number of controls, combined with the high inter- and intra-individual variability of steroid hormone concentrations, means that the control set may not fully represent the population as a whole. Furthermore, while those controls were selected to be as closely matched to the patient as possible, it would have been preferable to recruit volunteers specifically for this study had resources allowed.It seems extremely fortunate that in this case there was an error that could be detected by the short-read alignment, since this inversion would otherwise have been missed. Perhaps it was all part of the same event, with the DNA break being repaired by gene conversion but with the homology leading to the central section being fully inverted with the copy-and-paste errors further out. With such high homology making errors more likely, however, it is impossible to be sure that it was not sheer coincidence.Long-read sequencing has the power to identify and fully characterise structural variants in difficult-to-align regions, although the challenges that these regions present to algorithmic analysis mean that manual oversight of the bioinformatic pipeline is currently needed to ensure that variants are accurately identified. In particular, reads crossing SV breakpoints may be aligned intact rather than split, instead showing clusters of SNVs and/or mid-scale indels that may confound SV callers but are easily seen visually. Improved detection of variants in these regions has the potential to provide a molecular diagnosis for more of the many patients with clinically diagnosed genetic disorders, and to reveal the underlying causes of conditions of unknown aetiology. We hope that as costs fall and algorithms improve, long-read methodologies will be widely adopted in clinical practice and research.Genetic variants causing upregulation of AKR1C2 combined with partial loss of AKR1C1 may constitute a novel pathology that underlies some cases of unexplained chronic fatigue. If they do then the biomarkers discovered in this study might allow better targeting of a specific patient subgroup for research and treatment. Genetic screening could be implemented, a serum steroid profile that reports the relevant ratios could be established as a simple diagnostic test, and clinical trials of UDCA, Sepranolone or Golexanolone could be conducted. Follow up studies with transcriptional and functional analyses of the inversion are recommended, along with genetic and biochemical assessment of other patients to determine if this is the case.Additional file 1. Genomic DNA by Ligation protocol.Additional file 2. PromethION SV calling pipeline.Additional file 3: Fig. S1. The cleanly phased BAM files aligned to consensus and inverted reference\u00a0sequences. In both panels, the upper alignments track is the ALT allele and the lower track is the REF allele. The inverted region is shown by the red bar. In panel a the alleles are aligned to the GRCh38 consensus sequence; the ALT allele shows signs of a large inversion while the REF allele fits well. In panel b the reads are aligned to the new reference, where the region between the proposed breakpoints is inverted; now the ALT allele fits well while the REF allele shows the signs of a large inversion. Fig. S2. The raw reads from the phased ALT allele mapped to the assembled ALT allele. INDELs of\u2009\u2264\u20095 bp are hidden. There are very few mismatched bases and no loci where more than half of the reads have a mismatched base, showing that the assembly is accurate.Additional file 4: Further explanation of the lower 17-OHP/17,20\u03b1-diOHP ratio in the patient\u2019s first luteal sample."
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